Oxidation of compounds using genetically modified candida

ABSTRACT

An α-carboxyl-ω-hydroxyl fatty acid oxidized from a substantially pure  Candida  host cell, wherein the substantially pure  Candida  host cell is characterized by a first genetic modification class that comprises one or more genetic modifications that collectively or individually disrupt an alcohol dehydrogenase gene, and a second genetic modification class, wherein the second genetic modification class comprises an insertion of a first gene into the  Candida  host cell genome.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/084,230, entitled “OXIDATION OF COMPOUNDS USING GENETICALLY MODIFIED CANDIDA,” filed on Nov. 19, 2013, which is a divisional of U.S. patent application Ser. No. 12/775,306, entitled “OXIDATION OF COMPOUNDS USING GENETICALLY MODIFIED CANDIDA,” filed on May 6, 2010, which issued as U.S. Pat. No. 8,597,923 on Dec. 3, 2013, which claims priority to U.S. Provisional Patent Application No. 61/176,064, entitled “BIOSYNTHETIC ROUTES TO ENERGY RICH MOLECULES USING GENETICALLY MODIFIED CANDIDA,” filed May 6, 2009, and which is a continuation-in-part of U.S. patent application Ser. No. 12/436,729, entitled “BIOSYNTHETIC ROUTES TO LONG-CHAIN ALPHA, OMEGA-HYDROXYACIDS, DIACIDS AND THEIR CONVERSION TO OLIGOMERS AND POLYMERS,” filed May 6, 2009, which issued as U.S. Pat. No. 8,158,391 on Apr. 17, 2012. The disclosures of the above-referenced applications are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number DAAD19-03-1-0091, W911QY-04-C-0082 and NBCH1070004 awarded by the Defense Advanced Research Projects Agency (DARPA) to Richard A. Gross. The United States Government has certain rights in this invention.

SEQUENCE LISTING

This application includes a Sequence Listing submitted as filename 49840.054DV1_SL.txt, of size 337,998 bytes, created Jun. 11, 2014. The Sequence Listing is incorporated by reference herein in its entirety.

1. FIELD

Methods for biological production of α,ω-hydroxyacids using genetically modified strains of the yeast Candida are provided. Also provided are methods for the genetic modification of the yeast Candida. Also provided are DNA constructs for removal of genes that can interfere with the production of energy rich molecules by Candida. Also provided are DNA constructs for insertion of genes for expression into the Candida genome.

2. BACKGROUND

Genes that encode proteins that catalyze chemical transformations of alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols may aid in the biosynthesis of energy rich molecules, or in the conversion of such compounds to compounds better suited to specific applications. Such molecules include hydrocarbons (alkane, alkene and isoprenoid), fatty acids, fatty alcohols, fatty aldehydes, esters, ethers, lipids, triglycerides, and waxes, and can be produced from plant derived substrates, such as plant cell walls (lignocellulose, cellulose, hemicellulose, and pectin) starch, and sugar. These molecules are of particular interest as potential sources of energy from biological sources, and thus as possible replacements from energy sources derived from crude oil and its distillates. These molecules are also of interest as potential sources of chemical intermediates, and thus as possible replacements for chemicals derived from crude oil and its distillates.

Yeasts from the genus Candida are industrially important, they tolerate high concentrations of fatty acids and hydrocarbons in their growth media and have been used to produce long chain fatty diacids (Picataggio et al. (1992), Biotechnology (N Y): 10, 894-8.) However they frequently lack enzymes that would facilitate conversion of plant cell wall material (cellulose, hemicellulose, pectins and lignins) into sugar monomers for use in biofuel production. Methods for addition of genes encoding proteins capable of catalyzing such conversion into the Candida genome are thus of commercial interest. Further, because yeasts do not always contain enzymatic systems for uptake and metabolism of all of the sugar monomers derived from plant cell wall material, genes encoding enzymes that enable Candida to utilize sugars that it does not normally use, and methods for adding these genes to the Candida genome, are thus of commercial interest.

Currently, α,ω-dicarboxylic acids are almost exclusively produced by chemical conversion processes. However, the chemical processes for production of α,ω-dicarboxylic acids from non-renewable petrochemical feedstocks usually produces numerous unwanted byproducts, requires extensive purification and gives low yields (Picataggio et al., 1992, Bio/Technology 10, 894-898). Moreover, α,ω-dicarboxylic acids with carbon chain lengths greater than 13 are not readily available by chemical synthesis. While several chemical routes to synthesize long-chain α,ω-dicarboxylic acids are available, their synthesis is difficult, costly and requires toxic reagents. Furthermore, most methods result in mixtures containing shorter chain lengths. Furthermore, other than four-carbon α,ω-unsaturated diacids (e.g. maleic acid and fumaric acid), longer chain unsaturated α,ω-dicarboxylic acids or those with other functional groups are currently unavailable since chemical oxidation cleaves unsaturated bonds or modifies them resulting in cis-trans isomerization and other by-products.

Many microorganisms have the ability to produce α,ω-dicarboxylic acids when cultured in n-alkanes and fatty acids, including Candida tropicalis, Candida cloacae, Cryptococcus neoforman and Corynebacterium sp. (Shiio et al., 1971, Agr. Biol. Chem. 35, 2033-2042; Hill et al., 1986, Appl. Microbiol. Biotech. 24: 168-174; and Broadway et al., 1993, J. Gen. Microbiol. 139, 1337-1344). Candida tropicalis and similar yeasts are known to produce α,ω-dicarboxylic acids with carbon lengths from C12 to C22 via an ω-oxidation pathway. The terminal methyl group of n-alkanes or fatty acids is first hydroxylated by a membrane-bound enzyme complex consisting of cytochrome P450 monooxygenase and associated NADPH cytochrome reductase that is the rate-limiting step in the ω-oxidation pathway. Two additional enzymes, the fatty alcohol oxidase and fatty aldehyde dehydrogenase, further oxidize the alcohol to create ω-aldehyde acid and then the corresponding α,ω-dicarboxylic acid (Eschenfeldt et al., 2003, Appl. Environ. Microbiol. 69, 5992-5999). However, there is also a β-oxidation pathway for fatty acid oxidation that exists within Candida tropicalis. Both fatty acids and α,ω-dicarboxylic acids in wild type Candida tropicalis are efficiently degraded after activation to the corresponding acyl-CoA ester through the β-oxidation pathway, leading to carbon-chain length shortening, which results in the low yields of α,ω-dicarboxylic acids and numerous by-products.

Mutants of C. tropicalis in which the β-oxidation of fatty acids is impaired may be used to improve the production of α,ω-dicarboxylic acids (Uemura et al., 1988, J. Am. Oil. Chem. Soc. 64, 1254-1257; and Yi et al., 1989, Appl. Microbiol. Biotech. 30, 327-331). Recently, genetically modified strains of the yeast Candida tropicalis have been developed to increase the production of α,ω-dicarboxylic acids. An engineered Candida tropicalis (Strain H5343, ATCC No. 20962) with the POX4 and POX5 genes that code for enzymes in the first step of fatty acid β-oxidation disrupted was generated so that it can prevent the strain from metabolizing fatty acids, which directs the metabolic flux toward ω-oxidation and results in the accumulation of α,ω-dicarboxylic acids (FIGS. 3A and 3B). See U.S. Pat. No. 5,254,466 and Picataggio et al., 1992, Bio/Technology 10: 894-898, each of which is hereby incorporated by reference herein. Furthermore, by introduction of multiple copies of cytochrome P450 and reductase genes into C. tropicalis in which the β-oxidation pathway is blocked, the C. tropicalis strain AR40 was generated with increased ω-hydroxylase activity and higher specific productivity of diacids from long-chain fatty acids. See, Picataggio et al., 1992, Bio/Technology 10: 894-898 (1992); and U.S. Pat. No. 5,620,878, each of which is hereby incorporated by reference herein. Genes encoding proteins that catalyze chemical transformations of alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols may also reduce the usefulness of these compounds as energy sources, for example by oxidizing them or further metabolizing them. Methods for identifying and eliminating from the Candida genome genes encoding enzymes that oxidize or metabolize alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols are thus of commercial interest. For example fatty alcohols cannot be prepared using any described strain of Candida because the hydroxy fatty acid is oxidized to form a dicarboxylic acid, which has reduced energy content relative to the hydroxy fatty acid. Furthermore, neither the general classes nor the specific sequences of the Candida enzymes responsible for the oxidation from hydroxy fatty acids to dicarboxylic acids have been identified. There is therefore a need in the art for methods to prevent the oxidation of hydroxy fatty acids to diacids during fermentative production.

3. SUMMARY

Methods for the genetic modification of Candida species to produce strains improved for the production of biofuels are disclosed. Methods by which yeast strains may be engineered by the addition or removal of genes to modify the oxidation of compounds of interest as biofuels are disclosed. Enzymes to facilitate conversion of plant cell wall material (cellulose, hemicellulose, pectins and lignins) into sugar monomers and enzymes to enable Candida to utilize such sugars for use in biofuel production and methods for addition of genes encoding such enzymes into the Candida genome are disclosed.

One embodiment provides a substantially pure Candida host cell for the production of an α-carboxyl-ω-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an α,ω-dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof. The Candida host cell is characterized by a first genetic modification class and a second genetic modification class. The first genetic modification class comprises one or more genetic modifications that disrupt the β-oxidation pathway in the substantially pure Candida host cell. The second genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one gene in the substantially pure Candida host cell selected from the group consisting of a CYP52A type cytochrome P450, a fatty alcohol oxidase, and an alcohol dehydrogenase.

Another embodiment provides a method for producing an α-carboxyl-ω-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an α,ω-dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof in a Candida host cell. The method comprises (A) making one or more first genetic modifications in a first genetic modification class to the Candida host cell. The method further comprises (B) making one or more second genetic modifications in a second genetic modification class to the Candida host cell, where steps (A) and (B) collectively form a genetically modified Candida host cell. The method further comprises (C) producing an α-carboxyl-ω-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an α,ω-dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof, by fermenting the genetically modified Candida host cell in a culture medium comprising a nitrogen source, an organic substrate having a carbon chain length in the range from C6 to C22, and a cosubstrate. Here, the first genetic modification class comprises one or more genetic modifications that disrupt the β-oxidation pathway of the Candida host cell. Also, the second genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one gene selected from the group consisting of a CYP52A type cytochrome P450, a fatty alcohol oxidase, and an alcohol dehydrogenase in the Candida host cell.

One embodiment provides a substantially pure Candida host cell for the production of energy rich molecules. The Candida host cell is characterized by a first genetic modification class and a second genetic modification class. The first genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one gene in the substantially pure Candida host cell selected from the group consisting of a fatty alcohol oxidase, and an alcohol dehydrogenase. The second genetic modification class comprises one or more genetic modifications that collectively or individually add to the host cell genome at least one gene selected from the group consisting of a lipase, a cellulase, a ligninase or a cytochrome P450 that is not identical to a naturally occurring counterpart gene in the Candida host cell; or a lipase, a cellulase, a ligninase or a cytochrome P450 that is expressed under control of a promoter other than the promoter that controls expression of the naturally occurring counterpart gene in the Candida host cell.

One embodiment provides a substantially pure Candida host cell for the biotransformation of organic molecules. The Candida host cell is characterized by a first genetic modification class and a second genetic modification class. The first genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one alcohol dehydrogenase gene in the substantially pure Candida host cell. The second genetic modification class comprises one or more genetic modifications that collectively or individually add to the host cell genome at least one gene that is not identical to a naturally occurring counterpart gene in the Candida host cell; or at least one gene that is identical to a naturally occurring counterpart gene in the Candida host cell, but that is expressed under control of a promoter other than the promoter that controls expression of the naturally occurring counterpart gene in the Candida host cell.

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11 and ADH-B11B.

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose nucleotide sequence is

at least 95% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 90% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 85% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56,

or at least 80% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 75% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 70% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 65% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 60% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 50% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 40% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 30% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 20% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or

at least 10% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of any one of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a first peptide. In some embodiments the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments, the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163). It will be understood that in amino acid sequences presented herein, each “x” represents a placeholder for a residue of any of the naturally occurring amino acids.

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a second peptide. In some embodiments the second peptide has the sequence QYATADAVQAA (SEQ ID NO: 158). In some embodiments the second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164). In some embodiments the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a third peptide. In some embodiments the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide. In some embodiments the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments, the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide. In some embodiments the fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises at least one, two, three, four or five peptides selected from the group consisting of a first peptide having the sequence VKYSGVCH (SEQ ID NO: 156), a second peptide having the sequence QYATADAVQAA (SEQ ID NO: 158), a third peptide having the sequence CAGVTVYKALK (SEQ ID NO: 159), a fourth peptide having the sequence GQWVAISGA (SEQ ID NO: 160) and a fifth peptide having the sequence VGGHEGAGVVV (SEQ ID NO: 157).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it has at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous residues of any one of SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, or SEQ ID NO:155.

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, has at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a stretch of between 5 and 120 contiguous residues, between 40 and 100 contiguous residues, between 50 and 90 contiguous residues, between 60 and 80 contiguous residues of any one of SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, or SEQ ID NO:155.

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, has at least 90 percent sequence identity to a stretch of between 10 and 100 contiguous residues of any one of SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, or SEQ ID NO:155.

In some embodiments, the first genetic modification class causes disruption of an alcohol dehydrogenase in a Candida host cell. In some embodiments disruption of an alcohol dehydrogenase is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The rate of conversion of the substrate by the Candida host cell is compared with the rate of conversion produced by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.). The rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is deemed disrupted if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the Candida host cell than the second host cell.

In some embodiments, disruption of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The amount of the substrate converted to product by the Candida host cell in a specified time is compared with the amount of substrate converted to product by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.). The amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is deemed disrupted if the amount of product is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 25% lower in the Candida host cell than the second host cell.

In some embodiments, the first genetic modification class causes an alcohol dehydrogenases to have decreased function relative to the function of the wild-type counterpart in the Candida host cell.

In some embodiments, decreased function of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The rate of conversion of the substrate by the Candida host cell is compared with the rate of conversion produced by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.). The rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is deemed to have decreased function if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the Candida host cell than the second host cell

In some embodiments, decreased function of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The amount of the substrate converted to product by the Candida host cell in a specified time is compared with the amount of substrate converted to product by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.). The amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is deemed to have decreased function if the amount of product is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the Candida host cell than the second host cell.

In some embodiments, the first genetic modification class causes an alcohol dehydrogenases to have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.

In some embodiments, activity of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The rate of conversion of the substrate by the Candida host cell is compared with the rate of conversion produced by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.). The rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is deemed to have a modified activity spectrum if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, or at least 25% lower in the Candida host cell than the second host cell.

In some embodiments, activity of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The amount of the substrate converted to product by the Candida host cell in a specified time is compared with the amount of substrate converted to product by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.). The amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is deemed to have a modified activity spectrum if the amount of product is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower in the Candida host cell than the second host cell.

In some embodiments the second genetic modification class comprises addition of at least one modified CYP52A type cytochrome P450 selected from the group consisting of CYP52A13, CYP52A14, CYP52A17, CYP52A18, CYP52A12, and CYP52A12B.

Disclosed are biosynthetic routes that convert (oxidize) fatty acids to their corresponding α-carboxyl-ω-hydroxyl fatty acids. This is accomplished by culturing fatty acid substrates with a yeast, preferably a strain of Candida and more preferably a strain of Candida tropicalis. The yeast converts fatty acids to long-chain ω-hydroxy fatty acids and α,ω-dicarboxylic acids, and mixtures thereof. Methods by which yeast strains may be engineered by the addition or removal of genes to modify the oxidation products formed are disclosed. Fermentations are conducted in liquid media containing fatty acids as substrates. Biological conversion methods for these compounds use readily renewable resources such as fatty acids as starting materials rather than non-renewable petrochemicals For example, ω-hydroxy fatty acids and α,ω-dicarboxylic acids can be produced from inexpensive long-chain fatty acids, which are readily available from renewable agricultural and forest products such as soybean oil, corn oil and tallow. Moreover, a wide range of α-carboxyl-ω-hydroxyl fatty acids with different carbon length can be prepared because the biocatalyst accepts a wide range of fatty acid substrates. Products described herein produced by the biocatalytic methods described herein are new and not commercially available since chemical methods are impractical to prepare the compounds and biocatalytic methods to these products were previously unknown.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows two pathways for metabolism of fatty acids, ω-oxidation and β-oxidation, both of which exist in yeasts of the genus Candida including Candida tropicalis. The names of classes of compounds are shown, arrows indicate transformations from one compound to another, and the names of classes of enzymes that perform these conversions are indicated by underlined names adjacent to the arrows.

FIG. 2 shows two pathways for metabolism of fatty acids, ω-oxidation and β-oxidation, both of which exist in yeasts of the genus Candida including Candida tropicalis. The names of classes of compounds are shown, arrows indicate transformations from one compound to another, and the names of classes of enzymes that perform these conversions are indicated by underlined names adjacent to the arrows. By inactivating the genes encoding acyl coA oxidase (pox4 and pox5), the β-oxidation pathway is blocked (indicated by broken arrows), so that fatty acids are not used as substrates for growth. This genetic modification allows Candida species of yeast including Candida tropicalis to be used as a biocatalyst for the production of α,ω-diacids. See, for example, Picataggio et al., 1991, Mol Cell Biol 11, 4333-4339; and Picataggio et al., 1992, Biotechnology 10, 894-898. The β-oxidation pathway may be disrupted by any genetic modification or treatment of the host cells with a chemical for example an inhibitor that substantially reduces or eliminates the activity of one or more enzymes in the β-oxidation pathway, including the hydratase, dehydrogenase or thiolase enzymes, and thereby reduces the flux through that pathway and thus the utilization of fatty acids as growth substrates.

FIGS. 3A and 3B show an alignment, using ClustalW, of the amino acid sequences of alcohol dehydrogenase proteins predicted from the sequences of genes from Candida albicans and Candida tropicalis. The genes from Candida tropicalis were isolated as partial genes by PCR with degenerate primers, so the nucleic acid sequences of the genes and the predicted amino acid sequences of the encoded proteins are incomplete. Amino acid sequences of the partial genes are predicted and provided: SEQ ID NO:155 (ADH-A4), SEQ ID NO:154 (ADH-B4), SEQ ID NO:152 (ADH-A10), SEQ ID NO:153 (ADH-A10B) and SEQ ID NO:151 (ADH-B11).

FIG. 4 shows a schematic representation of a DNA “genomic targeting” construct for deleting sequences from the genome of yeasts. The general structure is that the construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast chromosome. Between these targeting sequences are two sites recognized by a site-specific recombinase (indicated as “recombinase site”). Between the two site specific recombinase sites are sequence elements, one of which encodes a selective marker and the other of which (optionally) encodes the site-specific recombinase that recognizes the recombinase sites. In one embodiment the sequences of the DNA construct between the targeting sequences is the “SAT1 flipper”, a DNA construct for inserting and deleting sequences into the chromosome of Candida (Reuss et al., (2004), Gene: 341, 119-27.). In the “SAT1 flipper”, the recombinase is the flp recombinase from Saccharomyces cerevisiae (Vetter et al., 1983, Proc Natl Acad Sci USA: 80, 7284-78) (FLP) and the flanking sequences recognized by the recombinase are recognition sites for the flp recombinase (FRT). The selective marker is the gene encoding resistance to the Nourseothricin resistance marker from transposon Tn1825 (Tietze et al., 1988, J Basic Microbiol: 28, 129-36.). The DNA sequence of the SAT1-flipper is given as SEQ ID NO: 1. The genomic targeting sequence can be propagated in bacteria, for example E coli, in which case the complete plasmid will also contain sequences required for propagation in bacteria, comprising a bacterial origin of replication and a bacterial selective marker such as a gene conferring antibiotic resistance. The targeting construct can be released from this plasmid in a linear form by digestion with one or more restriction enzymes with recognition sites that flank the targeting sequences.

FIG. 5 shows a schematic representation of the homologous recombination between a “genomic targeting” construct of the form shown in FIG. 4, with the DNA contained in a yeast genome (either in the chromosome or in the mitochondrial DNA). The targeting construct (A) contains two regions of sequence homology to the genomic sequence (B); the corresponding sequences in the genomic sequence flank the DNA region to be replaced. Introduction of the targeting construct into the host cell is followed by homologous recombination catalyzed by host cell enzymes. The result is an integrant of the targeting construct into the genomic DNA (C) and the excised DNA (D) which will generally be lost from the cell.

FIG. 6 shows a schematic representation of excision of the targeting construct from the yeast genome that occurs when expression of the recombinase in the targeting construct is induced in the integrant (A) shown in FIG. 5. Induction of the site-specific recombinase causes recombination between the two recombinase recognition sites. The result is the excision of the sequences between the two recombinase sites (C) leaving a single recombinase site in the genomic DNA (B).

FIG. 7 shows a schematic representation of a DNA “genomic targeting” construct for inserting sequences into the genome of yeasts. The general structure is that the construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast chromosome. Between these targeting sequences are two sites recognized by a site-specific recombinase (indicated as “recombinase site”). Between the two site specific recombinase sites are sequence elements, one of which encodes a selective marker and the other of which (optionally) encodes the site-specific recombinase that recognizes the recombinase sites. Insertion of additional sequences between one of the targeting sequences and its closest recombinase recognition site will result in those sequences being inserted into the chromosome after excision of the targeting construct (“Insertion sequences”). The genomic targeting sequence can be propagated in bacteria, for example E coli, in which case the complete plasmid will also contain sequences required for propagation in bacteria, comprising a bacterial origin of replication and a bacterial selective marker such as a gene conferring antibiotic resistance. The targeting construct can be released from this plasmid in a linear form by digestion with one or more restriction enzymes with recognition sites that flank the targeting sequences.

FIG. 8 shows a schematic representation of the homologous recombination between a “genomic targeting” construct of the form shown in FIG. 7, with the DNA contained in a yeast genome (either in the chromosome or in the mitochondrial DNA). The targeting construct (A) contains two regions of sequence homology to the genomic sequence (B); the corresponding sequences in the genomic sequence flank the DNA region to be replaced. Introduction of the targeting construct into the host cell is followed by homologous recombination catalyzed by host cell enzymes. The result is an integrant of the targeting construct into the genomic DNA (C) and the excised DNA (D) which will generally be lost from the cell.

FIG. 9 shows a schematic representation of excision of the targeting construct from the yeast genome that occurs when expression of the recombinase in the targeting construct is induced in the integrant (A) shown in FIG. 8. Induction of the site-specific recombinase causes recombination between the two recombinase recognition sites. The result is the excision of the sequences between the two recombinase sites (C) leaving a single recombinase site together with the additional sequences that were included between the targeting sequences and the recombinase site (see FIG. 7) in the genomic DNA (B).

FIG. 10 shows a schematic representation of three stages in generation of a targeted deletion in a yeast genome (either in the chromosome or in the mitochondrial DNA), and the results of a PCR test to distinguish between the three stages. (A) PCR primers (thick arrows) are designed to flank the targeted region. (B) Insertion of a genomic targeting construct into the genome inserts two recombinase sites, a recombinase gene and a selection marker between the two target sequences. This changes the size of the DNA segment between the two PCR primers; in the case shown the size is increased. (C) Induction of the recombinase results in excision of the recombinase encoding gene, the selective marker and one of the recombinase sites. This again changes the size of the DNA segment between the two PCR primers. (D) PCR amplification from yeast genomic DNA unmodified (gel lanes marked A), with integrated genomic targeting vector (gel lanes marked B) or after excision of the genomic targeting vector (gel lanes marked C).

FIG. 11 shows a schematic representation of a DNA “genomic targeting” construct for inserting or deleting sequences in the genome of yeasts. The general structure is that the construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast chromosome. Between these targeting sequences is a sequence that encodes a selective marker.

FIG. 12 shows two pathways for metabolism of fatty acids, ω-oxidation and β-oxidation, both of which exist in Candida species of yeast including Candida tropicalis. The names of classes of compounds are shown, arrows indicate transformations from one compound to another, and the names of classes of enzymes that perform these conversions are indicated by underlined names adjacent to the arrows. By inactivating the Candida tropicalis genes pox4 and pox5 (or their functional homologs in other Candida species), the β-oxidation pathway is blocked (indicated by broken arrows), so that fatty acids are not used as substrates for growth. Furthermore, inactivation of CYP52A type cytochrome P450 enzymes, as illustrated in the Figure, prevents the ω-oxidation of these fatty acids. These enzymes may also be responsible for some or all of the transformations involved in oxidizing ω-hydroxy fatty acids to α,ω-dicarboxylic acids. See Eschenfeldt et al., 2003, “Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis.” Appli. Environ. Microbiol. 69: 5992-5999, which is hereby incorporated by reference herein.

FIG. 13 shows the levels of ω-hydroxy myristate and the over-oxidized C14 diacid produced by Candida tropicalis strains DP1 (ura3A/ura3B pox5A::ura3A/pox5B::ura3A pox4A::ura3A/pox4B::URA3A) and DP174 (ura3A/ura3B pox5A::ura3A/pox5B::ura3A pox4A::ura3A/pox4B::URA3A ΔCYP52A17/ΔCYP52A18 ΔCYP52A13/ΔCYP52A14). Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/l, KH₂PO₄ 9.3 g/1) plus 30 g/l glucose. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 60 g/l glucose in a 125 ml flask, and grown at 30° C. and 250 rpm for 12 hours before addition of substrate. After addition of substrates growth was continued at 30° C. and 250 rpm. Parts A and B: the substrate methyl myristate was then added to a final concentration of 10 g/l and the pH was adjusted to between 7.5 and 8. The culture was pH controlled by adding 2 mol/l NaOH every 12 hours and glucose was fed as a cosubstrate by adding 400 g/l glucose every 8 hours. Samples were taken at the times indicated, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy myristate and of the C14 diacid produced by oxidation of the ω-hydroxy myristate were measured by LC-MS (liquid chromatography mass spectroscopy). The diacid was quantified relative to a known standard. No such standard was available for the ω-hydroxy myristate, so it was quantified by measuring the area under the peak in the MS chromatogram. Parts C and D: the substrates methyl myristate, sodium myristate or myristic acid were added to a final concentration of 10 g/l and the pH was adjusted to between 7.5 and 8. The culture was pH controlled by adding 2 mol/l NaOH every 12 hours and glucose was fed as a cosubstrate by adding 400 g/l glucose every 8 hours. Samples were taken after 48 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy myristate and of the C14 diacid produced by oxidation of the ω-hydroxy myristate were measured by LC-MS (liquid chromatography mass spectroscopy).

FIG. 14 shows two pathways for metabolism of fatty acids, ω-oxidation and β-oxidation, both of which exist in Candida species of yeast including Candida tropicalis. The names of classes of compounds are shown, arrows indicate transformations from one compound to another, and the names of classes of enzymes that perform these conversions are indicated by underlined names adjacent to the arrows. By inactivating the Candida tropicalis genes pox4 and pox5 (or their functional homologs in other Candida species), the β-oxidation pathway is blocked (indicated by broken arrows), so that fatty acids are not used as substrates for growth. Furthermore, inactivation of CYP52A type cytochrome P450 enzymes prevents the ω-oxidation of fatty acids. Several enzymes including, but not limited to CYP52A type P450s, are responsible for transformations involved in oxidizing ω-hydroxy fatty acids to α,ω-dicarboxylic acids. If other enzymes involved in oxidation of ω-hydroxy fatty acids are present in the strain, then the strain will convert ω-hydroxy fatty acids fed in the media to α,ω-dicarboxylic acids. If other enzymes involved in oxidation of ω-hydroxy fatty acids have been eliminated from the strain, then the strain will convert ω-hydroxy fatty acids fed in the media to α,ω-dicarboxylic acids.

FIG. 15 shows the levels of α,ω-dicarboxylic acids produced by Candida tropicalis strains DP186, DP258 and DP259 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g/l glycerol in a 125 ml flask, and grown at 30° C. and 250 rpm for 12 hours before addition of substrate. After addition of substrates growth was continued at 30° C. and 250 rpm. Part A: the substrate ω-hydroxy laurate was then added to a final concentration of 5 g/l and the pH was adjusted to between 7.5 and 8. Samples were taken after 24 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-dicarboxy laurate were measured by LC-MS (liquid chromatography mass spectroscopy). Part B: the substrate ω-hydroxy palmitate was then added to a final concentration of 5 g/l and the pH was adjusted to between 7.5 and 8. Samples were taken after 24 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-dicarboxy laurate were measured by LC-MS (liquid chromatography mass spectroscopy).

FIG. 16 shows the levels of α,ω-dicarboxylic acids produced by Candida tropicalis strains DP186, DP283 and DP284 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g/l glycerol in a 125 ml flask, and grown at 30° C. and 250 rpm for 12 hours before addition of substrate. After addition of substrates growth was continued at 30° C. and 250 rpm. Part A: the substrate ω-hydroxy laurate was then added to a final concentration of 5 g/l and the pH was adjusted to between 7.5 and 8. Samples were taken after 24 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-dicarboxy laurate were measured by LC-MS (liquid chromatography mass spectroscopy). Part B: the substrate ω-hydroxy palmitate was then added to a final concentration of 5 g/l and the pH was adjusted to between 7.5 and 8. Samples were taken after 24 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-dicarboxy laurate were measured by LC-MS (liquid chromatography mass spectroscopy).

FIG. 17 shows a phylogenetic tree with five Candida tropicalis alcohol dehydrogenase sequences (A10, B11, B2, A4 and B4) and two alcohol dehydrogenases from Candida albicans (Ca_ADH1A and Ca_ADH2A).

FIG. 18 shows a schematic design for selecting two sets of nested targeting sequences for the deletion of two alleles of a gene whose sequences are very similar, for example the alcohol dehydrogenase genes. The construct for the first allele uses ˜200 base pair at the 5′ end and ˜200 base pair at the 3′ end as targeting sequences (5′-ADH Out and 3′-ADH Out). The construct for the second allele uses two sections of ˜200 base pair between the first two targeting sequences (5′-ADH In and 3′-ADH In). These sequences are eliminated by the first targeting construct from the first allele of the gene and will thus serve as a targeting sequence for the second allele of the gene.

FIG. 19 shows the levels of α,ω-dicarboxylic acids produced by Candida tropicalis strains DP1, DP283 and DP415 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 30° C. and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 18 hours the preculture was diluted in fresh media to A₆₀₀=1.0. This culture was shaken until the A₆₀₀ reached between 5.0 and 6.0. Biocatalytic conversion was initiated by adding 5 ml culture to a 125 ml flask together with 50 mg of ω-hydroxy lauric acid, and pH adjusted to ˜7.5 with 2M NaOH. Part A: cell growth was followed by measuring the A₆₀₀ every 2 hours. Part B: formation of diacid; every 2 hours a sample of the cell culture was taken, acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-dicarboxy laurate were measured by LC-MS (liquid chromatography mass spectroscopy).

FIG. 20 shows the levels of α,ω-dicarboxylic acids produced by Candida tropicalis strains DP1, DP390, DP415, DP417, DP421, DP423, DP434 and DP436 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 30° C. and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/l, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 18 hours the preculture was diluted in fresh media to A₆₀₀=1.0. This culture was shaken until the A₆₀₀ reached between 5.0 and 6.0. Biocatalytic conversion was initiated by adding 5 ml culture to a 125 ml flask together with 50 mg of ω-hydroxy lauric acid, and pH adjusted to ˜7.5 with 2M NaOH. Formation of diacid was measured at the indicated intervals by taking a sample of the cell culture and acidifying to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-dicarboxy laurate were measured by LC-MS (liquid chromatography mass spectroscopy).

FIG. 21 shows a schematic representation of a DNA “genomic insertion” construct for inserting sequences to be expressed into the genome of yeasts. The general structure is that the construct has a gene for expression which is preceded by a promoter that is active in the yeast (Promoter 1). Promoter 1 comprises a linearization position which may be a site recognized by a restriction enzyme which cleaves the genomic insertion construct once to linearize it, or an annealing site for PCR primers to amplify a linear molecule from the construct. Three positions (A, B and C) are marked in Promoter 1 for reference in FIG. 22 when the construct is linearized. The gene for expression is optionally followed by a transcription terminator (Transcription terminator 1). The genomic insertion construct also comprises a selectable marker. The selectable marker is preferably one that is active in both bacterial and yeast hosts. To achieve this, the selectable marker may be preceded by a yeast promoter (promoter 2) and a bacterial promoter, and optionally it may be followed by a transcription terminator (transcription terminator 2). The genomic insertion construct also comprises a bacterial origin of replication.

FIG. 22 shows a schematic representation of the integration of a DNA “genomic insertion” construct into the DNA of a yeast genome. Part A shows an integration construct of the structure shown in FIG. 22, with parts marked. The construct is linearized, for example by digesting with an enzyme that recognizes a unique restriction site within promoter 1, or by PCR amplification, or by any other method, so that a portion of promoter 1 is at one end of the linearized construct (5′ part), and the remainder at the other end (3′ end). Three positions (A, B and C) are marked in Promoter 1, these refer to the positions in FIG. 21. Part B shows the intact Promoter 1 in the yeast genome, followed by the gene that is normally transcribed from Promoter 1 (genomic gene expressed from promoter 1). Three positions (A, B and C) are also marked in the genomic copy of Promoter 1. Part C shows the genome after integration of the construct. The construct integrates at position B in Promoter 1, the site at which the construct was linearized. This results in a duplication of promoter 1 in the genome, with one copy of the promoter driving transcription of the introduced gene for expression and the other copy driving the transcription of the genomic gene expressed from promoter 1.

FIG. 23 shows a specific embodiment of the DNA “genomic insertion” construct shown in FIG. 21. The general structure is that the construct has a gene for expression which is preceded by a promoter that is active in the yeast (the Candida tropicalis isocitrate lyase promoter). The isocitrate lyase promoter comprises a unique BsiWI site whereby the construct may be cleaved by endocunclease BsiWI once to linearize it. The gene for expression is followed by a transcription terminator (isocitrate lyase transcription terminator). The genomic insertion construct also comprises a selectable marker conferring resistance to the antibiotic zeocin. This selectable marker is active in both bacterial and yeast hosts and preceded by a yeast promoter (the TEF1 promoter) and a Bacterial promoter (the EM7 promoter), and followed by a transcription terminator (the CYC1 transcription terminator 2). The genomic insertion construct also comprises a bacterial origin of replication (the pUC origin of replication).

FIG. 24 shows the levels of α,ω-dicarboxylic acids and ω-hydroxy fatty acids produced by Candida tropicalis strains dp1, dp201 and dp428 (see table 3 for genotypes). Cultures of the yeast strains were grown at 30° c. and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media f (media f is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, k₂hpo₄ 7.2 g/1, kh₂po₄ 9.3 g/l) plus 20 g/l glucose plus 5 g/l ethanol. After 18 hours 3 ml of preculture was added to 27 ml fresh media f plus 20 g/l glucose plus 5 g/l ethanol in a 500 ml flask, and grown at 30° c. and 250 rpm for 20 hours before addition of substrate. Biocatalytic conversion was initiated by adding 40 g/l of methyl myristate, the ph was adjusted to ˜7.8 with 2 m naoh. The culture was ph controlled by adding 2 mol/l naoh every 12 hours, glycerol was fed as cosubstrate by adding 500 g/l glycerol and ethanol was fed as a inducer by adding 50% ethanol every 12 hours. Samples were taken at the times indicated, cell culture was acidified to ph ˜1.0 by addition of 6 n hcl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy myristate and α,ω-dicarboxymyristate were measured by lc-ms (liquid chromatography mass spectroscopy).

FIG. 25 shows the levels of α,ω-dicarboxylic acids and ω-hydroxy fatty acids produced by Candida tropicalis strains dp428 and dp522 (see table 3 for genotypes). Cultures of the yeast strains were grown at 30° c. in a dasgip parallel fermentor containing 200 ml of media f (media f is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, k₂hpo₄ 7.2 g/1, kh₂po₄ 9.3 g/l) plus 30 g/l glucose. The ph was maintained at 6.0 by automatic addition of 6 m naoh or 2 m h₂so₄ solution. Dissolved oxygen was kept at 70% by agitation and o₂-cascade control mode. After 6 hour growth, ethanol was fed into the cell culture to 5 g/l. After 12 h growth, biocatalytic conversion was initiated by adding (a) 20 g/l of methyl myristate, (b) 20 g/l oleic acid or (c) 10 g/l linoleic acid. During the conversion phase, 80% glycerol was fed as ω-substrate for conversion of methyl myristate and 500 g/l glucose was fed as ω-substrate for conversion of oleic acid and linoleic acid by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%). Every 12 hour, ethanol was added into cell culture to 2 g/l, and fatty acid substrate was added to 20 g/l until the total substrate concentration added was (a) 60 g/l of methyl myristate, (b) 60 g/l oleic acid or (c) 30 g/l linoleic acid. Formation of products was measured at the indicated intervals by taking samples and acidifying to ph ˜1.0 by addition of 6 n hcl; products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy fatty acids and α,ω-dicarboxylic acids were measured by lc-ms (liquid chromatography mass spectroscopy).

FIG. 26 shows the levels of α,ω-dicarboxylic acids and ω-hydroxy fatty acids produced by Candida tropicalis strain dp428 (see table 3 for genotype) in two separate fermentor runs. C. Tropicalis dp428 was taken from a glycerol stock or fresh agar plate and inoculated into 500 ml shake flask containing 30 ml of ypd medium (20 g/l glucose, 20 g/l peptone and 10 g/l yeast extract) and shaken at 30° c., 250 rpm for 20 hours. Cells were collected by centrifugation and re-suspended in fm3 medium for inoculation. (fm3 medium is 30 g/l glucose, 7 g/l ammonium sulfate, 5.1 g/l potassium phosphate, monobasic, 0.5 g/l magnesium sulfate, 0.1 g/l calcium chloride, 0.06 g/l citric acid, 0.023 g/l ferric chloride, 0.0002 g/l biotin and 1 ml/l of a trace elements solution. The trace elements solution contains 0.9 g/l boric acid, 0.07 g/l cupric sulfate, 0.18 g/l potassium iodide, 0.36 g/l ferric chloride, 0.72 g/l manganese sulfate, 0.36 g/l sodium molybdate, 0.72 g/l zinc sulfate.) Conversion was performed by inoculating 15 ml of preculture into 135 ml fm3 medium, methyl myristate was added to 20 g/l and the temperature was kept at 30° c. The ph was maintained at 6.0 by automatic addition of 6 m naoh or 2 m h₂so₄ solution. Dissolved oxygen was kept at 70% by agitation and o₂-cascade control mode. After six hour growth, ethanol was fed into the cell culture to 5 g/l. During the conversion phase, 80% glycerol was fed as ω-substrate by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%). Every 12 hour, ethanol was added into cell culture to 2 g/l, and methyl myristate was added to 40 g/l until the total methyl myristate added was 140 g/l (e.g. the initial 20 g/l plus 3 subsequent 40 g/l additions). Formation of products was measured at the indicated intervals by taking samples and acidifying to ph ˜1.0 by addition of 6 n hcl; products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy myristate and α,ω-dicarboxymyristate were measured by lc-ms (liquid chromatography mass spectroscopy).

FIG. 27 shows the red fluorescent protein mCherry produced by Candida tropicalis strain DP197 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 30° C. on plates containing Buffered Minimal Medium+0.5% Glucose, 0.5% Glycerol, and 0.5% EtOH.

5. DETAILED DESCRIPTION

It is to be understood that what is disclosed herein is not limited to the particular methodology, devices, solutions or apparatuses described, as such methods, devices, solutions or apparatuses can, of course, vary.

5.1. DEFINITIONS

Use of the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of polynucleotides, reference to “a substrate” includes a plurality of such substrates, reference to “a variant” includes a plurality of variants, and the like.

Terms such as “connected,” “attached,” “linked,” and “conjugated” are used interchangeably herein and encompass direct as well as indirect connection, attachment, linkage or conjugation unless the context clearly dictates otherwise. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed in the disclosed embodiments. Where a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also encompassed. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosed embodiments. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any embodiment is disclosed as having a plurality of alternatives, examples of that embodiment in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosed embodiment can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, New York, 1994, and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y., 1991, provide one of ordinary skill in the art with a general dictionary of many of the terms used herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed embodiments, the preferred methods and materials are described. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The terms defined immediately below are more fully defined by reference to the specification as a whole.

As used, herein, computation of percent identity takes full weight of any insertions in two sequences for which percent identity is computed. To compute percent identity between two sequences, they are aligned and any necessary insertions in either sequence being compared are then made in accordance with sequence alignment algorithms known in the art. Then, the percent identity is computed, where each insertion in either sequence necessary to make the optimal alignment between the two sequences is counted as a mismatch.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” and “gene” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (“PNAs”)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and these terms are used interchangeably herein. These terms refer only to the primary structure of the molecule. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, and hybrids thereof including for example hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, “caps,” substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes (e.g. nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.

Where the polynucleotides are to be used to express encoded proteins, nucleotides that can perform that function or which can be modified (e.g., reverse transcribed) to perform that function are used. Where the polynucleotides are to be used in a scheme that requires that a complementary strand be formed to a given polynucleotide, nucleotides are used which permit such formation.

It will be appreciated that, as used herein, the terms “nucleoside” and “nucleotide” will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., where one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or is functionalized as ethers, amines, or the like.

Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the NI and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2, of cytidine and the C2-NH₂, N′—H and C6-oxy, respectively, of guanosine. Thus, for example, guanosine (2-amino-6-oxy-9-beta-D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9-beta-D-ribofuranosyl-purine). Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (1-beta-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-beta-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine (U.S. Pat. No. 5,681,702 to Collins et al., hereby incorporated by reference in its entirety). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine may be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides may be prepared using the method described by Switzer et al., 1993, supra, and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 to Collins et al., each of which is hereby incorporated by reference in its entirety. Other nonnatural base pairs may be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37, hereby incorporated by reference in it entirety, for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotidic units which form unique base pairs are known, such as those described in Leach et al., 1992, J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.

The phrase “DNA sequence” refers to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded, DNA or RNA, but double stranded DNA sequences are preferable. The sequence can be an oligonucleotide of 6 to 20 nucleotides in length to a full length genomic sequence of thousands or hundreds of thousands of base pairs. DNA sequences are written from 5′ to 3′ unless otherwise indicated.

The term “protein” refers to contiguous “amino acids” or amino acid “residues.” Typically, proteins have a function. However, for purposes of this disclosure, proteins also encompass polypeptides and smaller contiguous amino acid sequences that do not have a functional activity. The functional proteins of this disclosure include, but are not limited to, esterases, dehydrogenases, hydrolases, oxidoreductases, transferases, lyases, ligases, receptors, receptor ligands, cytokines, antibodies, immunomodulatory molecules, signaling molecules, fluorescent proteins and proteins with insecticidal or biocidal activities. Useful general classes of enzymes include, but are not limited to, proteases, cellulases, lipases, hemicellulases, laccases, amylases, glucoamylases, esterases, lactases, polygalacturonases, galactosidases, ligninases, oxidases, peroxidases, glucose isomerases, nitrilases, hydroxylases, polymerases and depolymerases. In addition to enzymes, the encoded proteins which can be used in this disclosure include, but are not limited to, transcription factors, antibodies, receptors, growth factors (any of the PDGFs, EGFs, FGFs, SCF, HGF, TGFs, TNFs, insulin, IGFs, LIFs, oncostatins, and CSFs), immunomodulators, peptide hormones, cytokines, integrins, interleukins, adhesion molecules, thrombomodulatory molecules, protease inhibitors, angiostatins, defensins, cluster of differentiation antigens, interferons, chemokines, antigens including those from infectious viruses and organisms, oncogene products, thrombopoietin, erythropoietin, tissue plasminogen activator, and any other biologically active protein which is desired for use in a clinical, diagnostic or veterinary setting. All of these proteins are well defined in the literature and are so defined herein. Also included are deletion mutants of such proteins, individual domains of such proteins, fusion proteins made from such proteins, and mixtures of such proteins; particularly useful are those which have increased half-lives and/or increased activity.

“Polypeptide” and “protein” are used interchangeably herein and include a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” “oligopeptides,” and “proteins” are included within the definition of polypeptide. The terms include polypeptides containing in co- and/or post-translational modifications of the polypeptide made in vivo or in vitro, for example, glycosylations, acetylations, phosphorylations, PEGylations and sulphations. In addition, protein fragments, analogs (including amino acids not encoded by the genetic code, e.g. homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), natural or artificial mutants or variants or combinations thereof, fusion proteins, derivatized residues (e.g. alkylation of amine groups, acetylations or esterifications of carboxyl groups) and the like are included within the meaning of polypeptide.

“Amino acids” or “amino acid residues” may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

The term “expression system” refers to any in vivo or in vitro biological system that is used to produce one or more protein encoded by a polynucleotide.

The term “translation” refers to the process by which a polypeptide is synthesized by a ribosome ‘reading’ the sequence of a polynucleotide.

In some embodiments, the term “disrupt” means to reduce or diminish the expression of a gene in a host cell organism.

In some embodiments, the term “disrupt” means to reduce or diminish a function of a protein encoded by a gene in a host cell organism. This function may be, for example, an enzymatic activity of the protein, a specific enzymatic activity of the protein, a protein-protein interaction that the protein undergoes in a host cell organism, or a protein-nucleic acid interaction that the protein undergoes in a host cell organism.

In some embodiments, the term “disrupt” means to eliminate the expression of a gene in a host cell organism.

In some embodiments, the term “disrupt” means to eliminate the function of a protein encoded by a gene in a host cell organism. This function may be, for example, an enzymatic activity of the protein, a specific enzymatic activity of the protein, a protein-protein interaction that the protein undergoes in a host cell organism, or a protein-nucleic acid interaction that the protein undergoes in a host cell organism.

In some embodiments, the term “disrupt” means to cause a protein encoded by a gene in a host cell organism to have a modified activity spectrum (e.g., reduced enzymatic activity) relative to wild-type activity spectrum of the protein.

In some embodiments, disruption is caused by mutating a gene in a host cell organism that encodes a protein. For example, a point mutation, an insertion mutation, a deletion mutation, or any combination of such mutations, can be used to disrupt the gene. In some embodiments, this mutation causes the protein encoded by the gene to express poorly or not at all in the host cell organism. In some embodiments, this mutation causes the gene to no longer be present in the host cell organism. In some embodiments, this mutation causes the gene to no longer encode a functional protein in the host cell organism. The mutation to the gene may be in the portion of the gene that encodes a protein product (exon), it may be in any of the regulatory sequences (e.g., promoter, enhancer, etc.) that regulate the expression of the gene, or it may arise in an intron.

In some embodiments, the disruption (e.g., mutation) of a gene causes the protein encoded by the gene to have a mutation that diminishes a function of the protein relative to the function of the wild type counterpart of the mutated protein.

As used, herein, the wild type counterpart of a mutated protein is the unmutated protein, occurring in wild type host cell organism, which corresponds to the mutated protein. For example, if the mutated protein is a protein encoded by mutated Candida tropicalis POX 5, the wild type counterpart of the mutated protein is the gene product from naturally occurring Candida tropicalis POX 5 that is not mutated.

As used herein, the wild type counterpart of a mutated gene is the unmutated gene occurring in wild type host cell organism, which corresponds to the mutated gene. For example, if the mutated gene is Candida tropicalis POX 5 containing a point mutation, the wild type counterpart is Candida tropicalis POX 5 without the point mutation.

In some embodiments, a gene is deemed to be disrupted when the gene is not capable of expressing protein in the host cell organism.

In some embodiments, a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 20% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).

In some embodiments, a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 30% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).

In some embodiments, a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 40% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).

In some embodiments, a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 50% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).

In some embodiments, a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 60% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).

In some embodiments, a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 70% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).

In some embodiments, a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 20% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).

In some embodiments, a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 30% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).

In some embodiments, a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 40% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).

In some embodiments, a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 50% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).

In some embodiments, a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 60% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).

In some embodiments, a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 70% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has an enzymatic activity that is 20% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has an enzymatic activity that is 30% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has an enzymatic activity that is 40% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has an enzymatic activity that is 50% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has an enzymatic activity that is 60% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has an enzymatic activity that is 70% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments enzymatic activity is defined as moles of substrate converted per unit time=rate×reaction volume. Enzymatic activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which are to be specified. The SI unit for enzyme activity is the katal, 1 katal=1 mol s-1.

In some embodiments enzymatic activity is expressed as an enzyme unit (EU)=1 30 μmol/min, where 1 U corresponds to 16.67 nanokatals. See Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1979), “Units of Enzyme Activity,” Eur. J. Biochem. 97: 319-320, which is hereby incorporated by reference herein.

In some embodiments, a protein is deemed to be disrupted when a sample of the disrupted protein “disrupted sample” having a purity of 50% weight per weight (w/w) or weight per volume (w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that is 20% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein “wild type sample” in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when a sample of the disrupted protein “disrupted sample” having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that is 30% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein “wild type sample” in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when a sample of the disrupted protein “disrupted sample” having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that is 40% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein “wild type sample” in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when a sample of the disrupted protein “disrupted sample” having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that is 50% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein “wild type sample” in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when a sample of the disrupted protein “disrupted sample” having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that is 60% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein “wild type sample” in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when a sample of the disrupted protein “disrupted sample” having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that is 70% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein “wild type sample” in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by an assay that measures the consumption of substrate or the production of product over time such as those disclosed in Schnell et al., 2006, Comptes Rendus Biologies 329, 51-61, which is hereby incorporated by reference herein.

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by an initial rate experiment. In such an assay, the protein (enzyme) is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is relatively free from complications such as back-reaction and enzyme degradation.

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by progress curve experiments. In such experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium.

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by transient kinetics experiments. In such experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period.

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction.

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by continuous assays, where the assay gives a continuous reading of activity, or discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

In some embodiments, the enzymatic activity or enzymatic specific activity is measured by a fluorometric assay (e.g., Bergmeyer, 1974, “Methods of Enzymatic Analysis”, Vol. 4, Academic Press, New York, N.Y., 2066-2072), a calorimetric assay (e.g., Todd and Gomez, 2001, Anal Biochem. 296, 179-187), a chemiluminescent assay, a light scattering assay, a radiometric assay, or a chromatrographic assay (e.g., Churchwella et al., 2005, Journal of Chromatography B 825, 134-143).

In some embodiments, a protein is deemed to be disrupted when the protein has a function whose performance is 20% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has a function whose performance is 30% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has a function whose performance is 40% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has a function whose performance is 50% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has a function whose performance is 60% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is deemed to be disrupted when the protein has a function whose performance is 70% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).

In some embodiments, a protein is disrupted by a genetic modification. In some embodiments, a protein is disrupted by exposure of a host cell to a chemical (e.g., an inhibitor that substantially reduces or eliminates the activity of the enzyme). In some embodiments, this compound satisfies the Lipinski's Rule of Five: 30 (i) not more than five hydrogen bond donors (e.g., OH and NH groups), (ii) not more than ten hydrogen bond acceptors (e.g. N and O), (iii) a molecular weight under 500 Daltons, and (iv) a Log P under 5. The “Rule of Five” is so called because three of the four criteria involve the number five. See, Lipinski, 1997, Adv. Drug Del. Rev. 23, 3, which is hereby incorporated herein by reference in its entirety.

In some embodiments, the invention relates to nucleic acids hybridized using conditions of low stringency (low stringency conditions). By way of example and not limitation, hybridization using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792): filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 mg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm 32P-labeled probe. Filters are incubated in hybridization mixture for 18-20 hours at 40° C., and then washed for 1.5 hour at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hour at 60° C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68° C. and reexposed to film. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).

In some embodiments, the invention relates to nucleic acids under conditions of moderate stringency (moderately stringent conditions). As used herein, conditions of moderate stringency (moderately stringent conditions), are as known to those having ordinary skill in the art. Such conditions are also defined by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, 1989, which is hereby incorporated by reference herein in its entirety. They include, for example, use of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50 percent formamide, 6×SSC at 42° C. (or other similar hybridization solution, or Stark's solution, in 50% formamide at 42° C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS. See also, Ausubel et al., eds., in the Current Protocols in Molecular Biology series of laboratory technique manuals, ©1987-1997, Current Protocols, ©1994-1997, John Wiley and Sons, Inc., hereby incorporated by reference herein in its entirety. The skilled artisan will recognize that the temperature, salt concentration, and chaotrope composition of hybridization and wash solutions can be adjusted as necessary according to factors such as the length and nucleotide base composition of the probe. Other conditions of moderate stringency that may be used are well known in the art.

In some embodiments, the invention relates to nucleic acids under conditions of high stringency (high stringent conditions). As used herein conditions of high stringency (high stringent conditions) are as known to those having ordinary skill in the art. By way of example and not limitation, procedures using such conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65 C in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65 C in prehybridization mixture containing 100 mg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filters is done at 37 C for one hour in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50 C for 45 minutes before autoradiography. Other conditions of high stringency that may be used are well known in the art.

As used herein, computation of percent identity takes full weight of any insertions in two sequences for which percent identity is computed. To compute percent identity between two sequences, they are aligned and any necessary insertions in either sequence being compared are then made in accordance with sequence alignment algorithms known in the art. Then, the percent identity is computed, where each insertion in either sequence necessary to make the optimal alignment between the two sequences is counted as a mismatch. Unless explicitly indicated otherwise, the percent identity of two sequences is the percent identity across the entire length of each of the sequences being compared, with gaps insertions processed as specified in this paragraph.

5.2. ENZYMES TO DERIVE AND UTILIZE SUGAR FROM PLANT CELL WALLS AND PLANT STARCHES

Many biofuel production pathways start from sugars which are expensive and compete, directly or indirectly, with food crops. Commercially advantageous production pathways are those that begin with cheaper raw materials such as agricultural by-products, or agricultural products that require minimal processing for example cell wall material.

In addition to naturally occurring enzymes, modified enzymes may be added into the host genome. For example enzymes may be altered by incorporating systematically varied sets of amino acid changes, with the resulting changes in phenotypes measured and used to identify sequence changes conferring improved function (see for example Liao et al., 2007, BMC Biotechnol 7:16; Ehren et al., 2008, Protein Eng Des Sel 21:699-707 and Heinzelman et al., 2009, Proc Natl Acad Sci USA 106: 5610-5615).

5.2.1. Enzymes for Cellulose, Hemicelluose, and Lignocellulose Degradation

Organisms capable of generating enzymes for the breakdown of cellulose, hemicellulose, and pectin include, Trichoderma viride, Fusarium oxysporium, Piptoporus betulinus, Penicillium echinulatum, Penicillium purpurogenum, Penicillium rubrum, Aspergillus niger, Aspergillus fumigatus, Aspergillus phoenicus, Sporotrichum thermophile, Scytalidium thermophillum, Clostridium straminisolvens, Thermonospora curvata, Rhodospirillum rubrum, Cellulomonas fimi, Clostridium stercorarium, Bacillus polymyxa, Bacillus coagulans, Pyrococcu furiosus, Acidothermus cellulolyticus, Saccharophagus degradans, Neurospora crass, Humicola fuscoatra, Chaectomium globosum, Thielavia terrestris-255, Mycelieopthra fergussi-246C, Aspergillus wentii, Aspergillus ornatus, Pleurotus florida, Pleurotus cornucopiae, Tramates versicolor, Bacteroides thetaiotaomicron, and Nectria catalinensis; see Kumar et al., 2008, J Ind Microbiol Biotechnol: 35, 377-91.

5.2.1.1. Cellulose

Cellulose is a homopolymeric compound composed of β-D-glucopyranose units, linked by a β-(1→4)-glycosidic bond and represents the most abundant polysaccharide in plant cell walls.

Trichoderma reesei is one of the prototypical cellulose metabolizing fungi. It encodes genes for 3 enzyme classes required for the degradation of cellulose to glucose. These are Exoglucanases or cellobiohydrolases (genes CBH1 and CBH2), Endoglucanases (genes EG1, EG2, EG3, EG5) and β-glucosidase (gene BGL1). Genes for these 3 classes of enzymes could be expressed and secreted from a modified C. tropicalis strain to allow it to generate glucose from cellulose.

Clostridium thermocellum is a prototypical cellulose degrading bacterium. It encodes numerous genes that form the cellulosome, a complex of enzymes used in the degradation of cellulose. Enzymes participate in the formation of the cellulosome include scaffoldin (cipA), cellulase (celJ), cellobiohydrolase (cbhA, celK, cello), xylanase (xynY, xynZ, xynA, xynU, xynC, xynD, XynB, XynV), endoglucanase (celH, celE, celS, celF, celN, celQ, celD, celB, celT, celG, celA), mannanase (manA), chitinase (chiA), lichenase (licB) and a protein with unknown function CseP (cseP).

Encoding all or a subset of the genes required to replicate the C. thermocellum cellulosome, component enzymes or engineered derivatives would be of utility in a Candida strain configured for cellulose degradation. There is emerging evidence that effective hydrolysis of cellulose requires a multicomponent system like the cellulosome that interacts with the substrate and the surface of the cell. Cellulosomes are nanomachines consisting cellulase catalytic modules, carbohydrate binding domains that lock into the substrate, and dockerins plus cohesions that serve to connect the catalytic and carbohydrate binding domains to the surface of the bacterial cell that is expressing the cellulosome.

5.2.1.2. Hemicellulose:

Hemicellulose is the second most abundant component of plant cell walls. Hemicelluloses are heterogeneous polymers built up by many different sugar monomers. In contrast, cellulose contains only anhydrous glucose. For instance, besides glucose, sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses contain most of the D-pentose sugars, and occasionally small amounts of L-sugars as well. Xylose is always the sugar monomer present in the largest amount, but mannuronic acid and galacturonic acid also tend to be present.

Hemicellulose degrading enzymes include the xylan degrading enzymes (endo-β-xylanase, α-glucuronidase, α-arabinofuranosidase, and β-xylosidase) and glucomannan degrading enzymes β-mannanase and β-mannosidase). Xylan is the predominant component of hemicellulose from hardwood and agricultural plants, like grasses and stray. Glucomannan is the dominant component of hemicellulose from hardwood.

Cellulose does not typically exist in nature by itself and so other enzymes are needed for effective biomass utilization. Xylanases hydrolyze the β-1,4-xylan linkage of hemicellulose to produce the pentose xylose. There are a large number of distinct xylanase protein families. Some fungi secrete xylanase isozymes: Trichoderme viride makes 13 and Asperigillus niger produces 15. Xylanases will be an increasing important component of hemicellulose utilization as an added enzyme or part of an integrated bioprocessing system produced in situ by a suitable organism. Xylanases would be of utility in a Candida strain configured for cellulose degradation.

5.2.1.3. Pectin:

Pectins are the third main structural polysaccharide of plant cell walls. Pectins are abundant in sugar beat pulp and fruits, e.g., citrus and apples, where it can form up to ½ the polymeric content of cell walls. The pectin backbone consists of homo-galacturonic acid regions and neutral sugar side chains from L-rhamnose, arabinose, galactose, and xylose. L-rhamnose residues in the backbone carry sidechains containing arabinose and galactose. Pectin degrading enzymes include pectin lyase, endo-polygalacturonase, α-arabinofuranosidase, α-galactosidase, polymethylgalacturonase, pectin depolymerase, pectinase, exopolygalacturanosidase hydrolase, α-L-Rhamnosidase, α-L-Arabinofuranosidase, polymethylgalacturonate lyase (pectin lyase), polygalacturonate lyase (pectate lyase), exopolygalacturonate lyase (pectate disaccharide-lyase). Pectinases would be of utility in a Candida strain configured for cellulose degradation

5.2.2. Biological Delignification

The white rot fungi are a diverse group of Basidiomycetes that are capable of completely degrading all the major components of plant cell walls, including cellulose, hemicellulose and lignin. Phanerochaete chrysosporium is a prototypical example that has recently been the focus of a genome sequencing and anotization project. See review of genome project and genes used in delignification (Kersten et al., 2007, Fungal Genet Biol: 44, 77-87.).

Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, and lignin. The carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin, by hydrogen and covalent bonds. Biomass comes in many different types, which may be grouped into four main categories: (1) wood residues (including sawmill and paper mill discards), (2) municipal paper waste, (3) agricultural residues (including corn stover and sugarcane bagasse), and (4) dedicated energy crops (which are mostly composed of fast growing tall, woody grasses). Fermentation of lignocellulosic biomass to ethanol is an attractive route to energy feedstocks that supplements the depleting stores of fossil fuels. Biomass is a carbon-neutral source of energy, since it comes from dead plants, which means that the combustion of ethanol produced from lignocelluloses will produce no net carbon dioxide in the earth's atmosphere. Also, biomass is readily available, and the fermentation of lignocelluloses provides an attractive way to dispose of many industrial and agricultural waste products. Finally, lignocellulosic biomass is a very renewable resource. Many of the dedicated energy crops can provide high-energy biomass, which may be harvested multiple times each year.

One barrier to the production of biofuels from biomass is that the sugars necessary for fermentation are trapped inside the lignocellulose. Lignocellulose has evolved to resist degradation and to confer hydrolytic stability and structural robustness to the cell walls of the plants. This robustness or “recalcitrance” is attributable to the crosslinking between the polysaccharides (cellulose and hemicellulose) and the lignin via ester and ether linkages. Ester linkages arise between oxidized sugars, the uronic acids, and the phenols and phenylpropanols functionalities of the lignin. To extract the fermentable sugars, one must first disconnect the celluloses from the lignin, and then acid-hydrolyze the newly freed celluloses to break them down into simple monosaccharides. Another challenge to biomass fermentation is the high percentage of pentoses in the hemicellulose, such as xylose, or wood sugar. Unlike hexoses, like glucose, pentoses are difficult to ferment. The problems presented by the lignin and hemicellulose fractions are the foci of much contemporary research.

Hundreds of sequences from P. chrysosporium are predicted to encode extracellular enzymes including many oxidative enzymes potentially involved in lignocellulose degradation, including peroxidases, copper radical oxidases, FAD-dependent oxidases, and multicopper oxidases. The oxidases and peroxidases are responsible for generating reactive and nonspecific free radicals that affect lignin degradation. Enzymes that accelerate the rate of lignocellulose degradation would be of utility in a Candida strain configured for cellulose degradation.

Large and complex families of cytochrome P450s, peroxidases, glycoside hydrolases, proteases, copper radical oxidases and multicopper oxidases are observed in the P. chrysosporium genome. Structurally related genes may encode proteins with subtle differences in functions, and such diversity may provide flexibility needed to change environmental conditions (pH, temperature, ionic strength), substrate composition and accessibility, and wood species. Alternatively, some of the genetic multiplicity may merely reflect redundancy.

Lignin peroxidases (LiP) and manganese peroxidases (MnP) have been the most intensively studied extracelluar enzymes of P. chrysosporium. Also, implicated in lignocelluose degradation are, copper radical oxidases (e.g., glyoxal oxidase, GLX), flavin and cytochrome enzymes such as, cellobiose dehydrogenase (CDH), glucose oxidases (glucose 1-oxidase and glucose 2-oxidase), aryl alcohol oxidases, veratryl alcohol oxidase, multicopper oxidases (mco1).

Proteases produced by P. chrysosporium may be involved in activation of cellulase activity. P. chrysosporium apparently does not code for laccases, which are used by other organisms for lignocellulose degradation.

Other lignocellulose degrading organisms include Pleurotus erygii (has a versatile peroxidase that exhibits both LiP and MnP activities), Cyathus sp., Streptomyces viridosporus T7A (the lignin peroxidase, LiP, has been studied in some detail), Phelebia tremellosus, Pleurotus florida, Peurotus cornucopiae, Pleurotus ostreatus, Trametes versicolor, Irpex lacteus, Ganoderma lucidum, Ganoderma applanatum, Coriolus versicolor, Aspergillus 2BNL1, Aspergillus 1AAL1, Lentinus edodes UEC 2019, Ceriporiopsis subvermispora, Panus conchatus.

5.2.3. Enzymes Needed for Utilization of Starch:

Enzymes for saccharification include α-amylases, β-amylases, γ-amylases, glucoamylase, maltogenase and pullanase.

5.3. POTENTIAL FEEDSTOCKS USED DIRECTLY OR FOLLOWING ENZYMATIC, PHYSICAL, CHEMICAL, AND OR MECHANICAL PRETREATMENT

Almost anything derived from the Kingdom Plantae, and more specifically anything containing, lingnocellulose, cellulose, hemicellulose, pectin, and/or starch can be used as a feedstock for the production of biofuels.

The heterogeneous structure of the lignin polymer renders it highly difficult to degrade. Lignin degradation occurs quite slowly in nature via the action of wood rot fungi that produce ligninases. These fungi and some bacteria recycle the carbon locked in woody plants taking years to digest a large tree. A major strategy for increasing availability of sugar polymers is to genetically decrease the lignin content of plants. Alfalfa lines downregulated in several steps of lignin biosynthesis were tested for sugar release during chemical saccharification with promising results. Plant with the lowest lignin compensated by making more carbohydrate. Moreover, the carbohydrate was more readily released with decreasing lignin. Sugars present were xylose, arabinose, glucose, and galactose that were representative of hemicellulosic and pectic cell wall polymers (Chen et al., 2007, Nat Biotechnol: 25, 759-61.).

5.3.1. Physical, Chemical, and/or Mechanical Lignocellulose Pre-treatments

Lignocellulosic substrates used by an engineered C. tropicalis strain may include one or more of the following pretreatments: mechanical pretreatment (milling), thermal pretreatment (steam pretreatment, steam explosion, and/or liquid hot water pretreatment), alkaline pretreatment, oxidative pretreatment, thermal pretreatment in combination with acid pretreatment, thermal pretreatment in combination with alkaline pretreatment, thermal pretreatment in combination with oxidative pretreatment, thermal pretreatment in combination with alkaline oxidative pretreatment, ammonia and carbon dioxide pretreatment, enzymatic pretreatment, and/or pretreatment with an engineered organism (Hendriks et al., 2009, Bioresour Technol: 100, 10-8.).

5.4. SUGARS DERIVED FROM PLANT CELL WALLS THAT MAY REQUIRE ENGINEERING OF C. TROPICALIS

Plant biomass hydrolysates contain carbon sources that may not be readily utilized by yeast unless appropriate enzymes are added via metabolic engineering (van Mans et al., 2006, Antonie Van Leeuwenhoek: 90, 391-418.). For example, S. cerivisiae readily ferments glucose, mannose, and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass will require metabolic engineering. The most abundant of these compounds is xylose. Other fermentable substrates include L-arabinose, galacturonic acid, and rhamnose.

5.4.1. Xylose Fermentation

Xylose-fermenting yeasts link xylose metabolism to the pentose-phosphate pathway. These yeasts use two oxidoreductases, xylose reductase (XR) and xylitol dehydrogenase (XDH), to convert xylose to xylulose 5-phosphate, which enters the pentose phosphate pathway.

Although strains of S. cerivisiae that express both xylose reductase (XR) and xylitol dehydrogenase (XDH) have been constructed, anaerobic fermentation was accompanied by considerable xylitol production. For every one NADPH used by XR, one NADH needs to be reoxidized, and the only way to do it be the engineered yeasts is to produce xylitol, although ethanol vs. xylitol production can be impacted both positively and negatively by starting strain, source of heterologous enzymes, and culture conditions. Ideally, the XR and XDH can be engineered to be linked to the same coenzyme system eliminating the production of excess NADH in the process of ethanol production.

One of the most successful examples of engineering S. cerivisiae for ethanol production from xylose uses the fungal xylose isomerase (Xy1A) from obligately anaerobic fungi Piromyces sp. E2. The introduction of the XylA gene was sufficient to enable the resulting strain to grow slowly with xylose as sole carbon source under aerobic conditions. Via an extensive selection procedure a new strain was derived (Kuyper et al., 2005, FEMS Yeast Res: 5, 399-409) which was capable of anaerobic growth on xylose producing mainly ethanol, CO2, glycerol, biomass, and notably little xylitol. The ethanol production rate was considered still too low for industrial applications. To obtain a higher specific rate of ethanol production, a strain was constructed that in addition to the Xy1A gene, overexpressed all genes involved in the conversion of xylose into the intermediates of glycolysis, including xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase, and transaldolase. In addition the gene GRE3, encoding aldose redcutase, was deleted to further minimize xylitol production. The resulting strain could be cultivated under anaerobic conditions without further selection or mutagenesis and at the time had the highest reported specific ethanol production rate.

Candida tropicalis has been shown to be able to ferment xylose to ethanol (Zhang et al., 2008, Sheng Wu Gong Cheng Xue Bao: 24, 950-6.) Pichia stipitis is another yeast that is able to ferment xylose to alcohol and being studied (Agbogbo et al., 2008, Appl Biochem Biotechnol: 145, 53-8).

5.4.2. L-Arabinose Fermentation

Although D-xylose is the most abundant pentose in hemicellulosic substrates, L-arabinose is present in significant amounts, thus the importance of converting arabinose to ethanol.

Saccharomyces cannot ferment or assimilate L-arabinose. Although many types of yeast are capable of assimilating L-arabinose aerobically, most are unable to ferment it to ethanol. Some Candida species are able to make arbinose fermentation to ethanol, but production rates are low.

L-arabinose fermentation may be-rare among yeasts due to a redox imbalance in the fungal L-arabinose pathway, therefore an alternative approach to using the fungal enzymes is to construct L-arabinose fermenting yeast by overexpression of the bacterial L-arabinose pathway. In the bacterial pathway no redox reactions are involved in the initial steps of L-arabinose metabolism. Instead the enzymes, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase are involved in converting L-arabinose to L-ribulose-5-phosphate and D-xyulose-5-phosphate, respectively. These enzymes are encoded by the araA, araB, and araD genes respectively.

A first attempt to express the E. coli genes in S. cerivisiae was only partly successful, with the strain generating only L-arabinitol. One of the most promising examples of S. cerivisiae engineering for L-arabinose fermentation is described in (Becker et al., 2003, Appl Environ Microbiol: 69, 4144-50.). In this work the bacterial L-arabinose operon consisted of E. coli araB and araD and Bacillus subtilis araA, along with overexpression of the yeast galactose permease gene (GAL2). Gal1p is known to transport L-arabinose. Although overexpression of these enzymes did not result in immediate growth on L-arabinose as the sole carbon source, the growth rate of the transformants increased progressively after 4-5 days incubation. Eventually an L-arabinose-utilizing strain was selected after several sequential transfers in L-arabinose medium. In addition to being able to grow aerobically on L-arabinose, the evolved strain produced ethanol from L-arabinose at 60% the theoretical yield under oxygen-limited conditions. An enhanced transaldolase (TAL1) activity was reported to enhance L-arabinose fermentation and overexpression of GAL2 was found not to be essential for growth on L-arabinose, suggesting that other yeast sugar transporters can also transport L-arabinose. A similar approach would be feasible in Candida, re-coding the genes to be better expressed in Candida, and to remove those codons that are non-canonical in Candida.

5.4.3. Galacturonic Acid fermentation:

Reduction of galacturonic acid to the same level of a hexose requires the input of two electron pairs, for instance via two NADH-dependent reduction steps. Galacturonic acid is a major component of pectin and therefore occurs in all plant biomass hydrolysates. Pectin-rich residues from citrus fruit, apples, sugar cane and sugar beets contain especially large amounts of D-galacturonic acid. If D-galacturonic acid can be converted to ethanol, this would increase the relevance of these abundantly available feedstocks.

Several yeasts, e.g., Candida and Pichia, can grow on D-galacturonic acid, and therefore potential sources for transport enzymes and a heterologous pathway if needed.

The ability to utilize D-galacturonic acid is widespread among bacteria, which all seem to use the same metabolic pathway. In the bacterial pathway, D-galacturonic acid is converted to pyruvate and glyceraldehydes-3-phosphate via a five-step pathway. Overall this results in the conversion of D-galacturonic acid, NADH, and ATP into pyruvate, glyceraldehydes-3-phosphate and water. Glyceraldehyde-3-phosphate can be converted to equimolar amounts of ethanol and CO2 via standard glycolytic reactions yielding 2 ATP. However, conversion of pyruvate to ethanol requires oxidation of a second NADH.

During anaerobic growth and fermentation on sugars (hexoses, but also xylose by engineered xylose-fermenting strains) of S. cerivisiae, a significant fraction of the carbon is channeled into glycerol to compensate for oxidative, NADH-generating reactions in biosynthesis.

In theory, introduction of the prokaryotic glacturonic acid fermentation route into yeast can create an alternative redox sink for the excess NADH formed in biosynthesis. This would have two advantages. Firstly, the NADH derived from biosynthetic processes can be used to increase ethanol yield on glacturonic acid to 2 mol ethanol per mol of glacturonic acid, as the pyruvate formed can now be converted to ethanol. Secondly, since the sugar requirements production for glycerol are reduced, the ethanol yield on sugars will increase.

Bacterial D-galacturonate catabolism uses the following enzymes: D-galacturonate isomerase, altronate oxidoreductase, altronate dehydratase, 2-dehydro-3-deoxygluconokinase, 2-keto-3-deoxy-6-phosphogluconate aldolase, glyceraldehydes-3-phosphate. Although a large number of yeasts and molds use galacturonic acid as carbon and energy for growth, knowledge of the underlying metabolic process is limited. At present, the prokaryotic pathway offers the most promising approach for engineering Candida for galacturonic acid metabolism.

5.4.4. L-Rhamnose fermentation:

The deoxyhexose L-rhamnose is named after the plant it was first isolated from: the buckthorn (Rhamnus). In contrast with most natural sugars, L-rhamnose is much more common than D-rhamnose. It occurs as part of the rhamnogalacturonan of pectin and hemicellulose. Being a 6-deoxy sugar, L-rhamnose is more reduced than the rapidly fermentable sugars glucose and fructose.

S. cerivisiae cannot grow on L-rhamonose. The metabolic engineering of S. cerivisiae for the production of ethanol will have to address two key aspects: the enhancement of rhamnose transport across the plasma membrane and the introduction of a rhamnose-metabolizing pathway.

Two possible strategies to engineer uptake follow. Firstly, after introduction of an ATP-yielding pathway for L-rhamnose catabolism (see below), selection for growth on L-rhamnose can be used to investigate whether or not mutations in hexose transporters enable uptake of L-rhamnose.

Although the rhamnose transporters from bacteria (e.g., E. coli) are well characterized, functional expression of bacterial transporters in the yeast plasma membrane may be challenging. Pichia stipidis is able to use L-rhamnose. Using information generated by the P. stipidis genome project, it might be possible to identify a rhamnose transporter if such a gene can be shown to be induced by rhamnose (as proposed for galacturonic acid above).

After uptake the next requirement for successful rhamnose fermentation is conversion into intermediates of central metabolism.

Two pathways for rhamnose utilization have been reported in microorganisms.

The first catabolic pathway involves phosphorylated intermediates and is used, for example, by E. coli. In this pathway, L-rhamnose is converted to L-rhamnulose by L-rhamnose isomerase. After the subsequent phosphorylation to L-rhamnulose by rhamnulokinase, L-rhamnulose-1-phosphate is split into dihdroxy-acetone-phosphate (DHAP) and L-lactaldehyde by rhamnulose-1-phosphate aldolase. DHAP can be normally processed by glycolysis, yielding 1 mol ethanol per mol L-rhamnose. In E. coli, further metabolism of L-lactaldehyde depends on the redox state of the cells. L-lactaldehyde can be oxidized to lactate by lactaldehyde dehydrogenase, reduced to 1,2-propanediol by lactaldehyde reductase, or processed via a redox-neutral mix of these two reactions. Introduction of this pathway into S. cerivisiae, L-rhamnulose is expected to be converted to equimolar amounts of ethanol, lactaldehyde and CO2 with generation of 1 ATP. In summary, this strategy would require the introduction of a transporter and three heterologous enzymes into S. cerivisiae.

A second route for rhamnose degradation, which does not involve phosphorylated intermediates was first described for the fungus Aureobasidium pullulans and is referred to as direct oxidative catabolism of rhamnose. A similar pathway occurs in the yeasts P. stipitis and Debaryomyces polymorphus. This pathway is initiated by the oxidation of L-rhamnose by NAD+-dependent L-rhamnose dehydrogenase, yielding either L-rhamnono-1,4-lactone or the unstable rhamnono-1,5-lactone. The 1,4 lactone is hydrolyzed to L-rhamnonate by L-rhamnono-1,4-lactonase. The unstable 1,5-lactone has been reported to spontaneously hydrolyze to L-rhamnonate. L-Rhamnonate is subsequently dehydrated to 2-keto-3-deoxy-L-rhamnonate by L-rhamnonate dehydratase. The product of this reaction is then cleaved into pyruvate and L-lactaldehyde by an aldolase. In P. stipitis the thus formed L-lactaldehyde is converted to lactate and NADH by lactaldehyde dehydrogenase. Introduction of this fungal pathway into S. cerivisiae should enable the conversion of L-rhamnose to equimolar amounts of ethanol, lactaldehyde and CO2 without a net generation of ATP. This conversion would require the introduction of a transporter and four heterologous enzymes (including 1,4-lactonase).

5.4.5. Inhibitor Tolerance:

The harsh conditions that prevail during the chemical and physical pretreatment of ligncullulse result in the release of many substances that inhibit growth and productivity of microorganisms such as S. cerivisiae. The number and identity of the toxic compounds varies with the nature of the raw material and pretreatment conditions.

There are two approaches to limit the impact of the inhibitors on the fermentation process: (i) introduction of additional chemical, physical, or biological process steps for removal or inactivation of inhibitors (ii) improvement of S. cerivisiae to the inhibitors.

5.5. FERMENTATION PRODUCTS FROM BIOMASS 5.5.1. Butanol

Metabolic engineering of Escherichia coli for butanol production by inserting genes from the butanol production bacteria Clostridium acetobutylicum into E. coli has been described (Inui et al., 2008, Appl Microbiol Biotechnol: 77, 1305-16).

A similar strategy can be envisioned for an engineered C. tropicalis strain configured to derive sugars from biomass. Enzymes (and genes) from Clostridium acetobutylicum required for butanol production from Acetyl-CoA include: Acetyl-CoA acetyltransferase (thiL), β-hydroxybutyryl-CoA dehydrogenase (hbd), 3-hydroxybutyryl-CoA dehydratase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), butyrlaldehyde dehydrogenase (adhe1, adhe), butanol dehydrogenase (adhe1, adhe), butyrlaldehyde dehydrogenase (bdhA), butanol dehydrogenase (bdhA), butyrlaldehde dehydrogenase (bdhB), butanol dehydrogenase (bdhB).

n-Butanol is a commercially important alcohol that is considered by some to be a strong Candidate for widespread use as a motor fuel. n-Butanol is currently produced via chemical synthesis almost exclusively. The dominant synthetic process in industry, the acetaldehyde method, relies on propylene derived from petroleum [1]. The U.S. market for butanol is 2.9 billion pounds per year [2]. Currently, the primary use of n-butanol is as a solvent, however, several companies including British Petroleum and DuPont are developing methods to utilize bacteria to produce n-butanol on a large scale for fuel [3]. Microorganisms capable of producing n-butanol by fermentation are Clostridia acetobutylicum, C. beijerinckii, and C. tetanomorphum.

n-Butanol has several characteristics that make it a viable alternative fuel option. It has an energy density that is similar to gasoline. Additionally, it could power a combustion engine with minimal or no modifications. In either a blended or neat form, n-butanol could be easily integrated into our current infrastructure.

Enzymes for butanol production include Pyruvate dehydrogenase complex, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, aldhyde and/or alcohol dehydrogenase. (Steen et al., 2008, Microb Cell Fact: 7, 36; Atsumi et al., 2008, Metab Eng: 10, 305-11.)

5.5.2. Branched Chain Alcohols

In the quest to find a substitute for petroleum based fuels, several low energy molecules have been suggested due to the ease of production. However, a molecule with similar energy density to current fuels would be preferred as a biofuel. Branched higher alcohols have a higher energy density than some of the alcohols proposed as alternative fuels. Other various properties of these alcohols also display more desirable features. For example, a lower miscibility with water and lower vapor pressure are benefits of higher alcohols. A unique approach to alcohol synthesis taken by Atsumi et al. Atsumi et al., 2008, Nature: 451, 86-9, employs synthetic biology to engineer non-fermentative pathways based on amino acid biosynthesis. These pathways produce alcohols that are not natural fermentation products. Some of the features of these molecules include branching and addition of aromatic cyclic hydrocarbon structures.

An engineered C. tropicalis capable of generating 2-methyl-1-butanol from L-threonine would use either the endogenous or exogenously added threonine biosynthetic enzymes, L-threonine ammonia lyase, endogenous or exogenously added isoluecine biosynthetic enzymes, 2-keto-acid decarboxylase, and an alcohol dehydrogenase.

3-methyl-1-butanol pathway from pyruvate would require valine biosynthesis enzymes, leucine biosynthesis enzymes, 2-keto-acid decarboxulase, alcohol dehydrogenase.

2-phenylethanol pathway from pyruvate would require Phenylalanine biosynthesis enzymes, 2-keto acid decarboxylase, alcohol dehydrogenase.

5.5.3. Isobutanol Pathway from Pyruvate

Isobutanol has a higher carbon content than ethanol, therefore making its energy properties closer to gasoline. Currently, isobutanol is used as a precursor for commodity chemicals including isobutyl acetate. Atsumi et al, 2008, Nature: 451, 86-9, synthesized isobutanol via synthetic biology. The origin of the enzymes required to synthesize isobutanol were from a variety of microorganisms including Lactococcus lactis and Saccharomyces cerevisiae. In addition to expressing foreign enzymes, the host, E. coli, was modified to direct metabolism toward isobutanol production. The interesting feature of this pathway to synthesize isobutanol is that it employs amino acid biosynthesis to generate the essential precursor. This allows the microbe to produce the alcohol in the presence of oxygen. In fact, semi-aerobic conditions increased yields. This approach has been applied to generate several other alcohols, such as 2-phenylethanol and 2-methyl-1-butanol. The pathways for these interesting alcohols have not yet been optimized.

Synthesis would require valine biosynthesis enzymes, 2-keto-acid decarboxylase, alcohol dehydrogenase.

5.5.4. Isopropanol Pathway from Pyruvate

Isopropanol is commonly employed as an industrial cleaner and solvent. Additionally, it is sold as “rubbing alcohol” for use as a disinfectant. As a significant component in dry gas, a fuel additive, it solubilizes water in gasoline, thereby removing the threat of frozen supply lines. Proposed biofuel applications include partial replacement of gasoline and in production of fatty acid esters. A benefit of substituting isopropanol for methanol in fatty acid esters is a higher tolerance for cold temperatures. The fatty acid isopropyl ester would remain liquid in cooler climates. The biosynthesis genes for isopropanol originally found in Clostridia acetobutylicum were engineered into an E. coli strain for optimal industrial usage (Hanai et al., 2007, Appl Environ Microbiol: 73, 7814-8).

Synthesis would require pyruvate dehydrogenase complex, acetyl-CoA acetyltransferase, acetoacetyltransferase, secondary alcohol dehydrogenase.

5.5.5. Methanol Pathway from Methane

Methanol can be synthesized chemically or biochemically from methane gas. Over 30 million tons per year of methanol are produced worldwide [1]. Currently, chemical synthesis is the method of choice. Methanol is widely used as a solvent, in antifreeze, and as an intermediate in synthesis of more complex chemicals. Methanol is used as a fuel in Indy race cars and it has been blended into gasoline for civilian automobiles. Microorganisms capable of methanol production include Methylobacterium sp., Methylococcus capsulatus, and Methylosinus trichosporium.

Enzymes required: methane monooxygenase.

5.5.6. Other Possible End Products Requiring Metabolic Engineering

Esters: Fatty acid ethyl ester, Fatty acid methyl ester Ethers: Dimethyl ether, Dimethylfuran, Methyl-t-butyl ether

Hydrocarbons: Alkanes, Alkenes, Isoprenoids 5.5.7. Over-Production of Fatty Acids

Because many of the strains described here are no longer able to utilize many fatty acids as carbon and energy sources due to the knockouts in both β-oxidation (pox4a/pox4b pox5a/pox5b) and ω-oxidation pathways (P450 (cytochrome P450), fao (fatty alcohol oxidase), and adh (alcohol dehydrogenase) gene), the strain is an ideal Candidate for metabolic engineering for manipulation of the fatty acid biosynthetic pathways for overproduction of fatty acids.

Fatty acids (and/or lipids) so produced could either be used for production of biofuels such as biodiesel or by restoring a P450 or P450s for endogenous production of ω-hydroxy fatty acids. Methods for over-production of endogenous fatty acids may be similar to those used by Lu X et al., 2008, Metab Eng: 10, 333-9.

Steps include:

1. Knocking out the E. coli fadD gene, which encodes an acyl-CoA synthetase, to block fatty acid degradation. This may be accomplished by knocking out acyl-coA synthetases and acyl-coA oxidases of the Candida tropicalis (e.g., POX4 and POX4 genes are already absent).

2. Heterologous expression of acyl-ACP thioesterases to increase the abundance of shorter chain fatty acids, e.g., U31813 from Cinnamomum camphorum (improved fuel quality).

3. Increasing the supply of malonyl-CoA by over-expressing acetyl-coA carboxylase.

4. Releasing feedback inhibition caused by long-chain fatty acids by overexpression of an endogenous or exogenous acyl-ACP thioesterase. Acyl-ACP thioesterases release free fatty acids from acyl-ACPs.

Mechanisms for membrane proliferation (more membrane=more lipid?):

Expression/overexpression of P450s including fatty acid, alkane, and alkene metabolizing P450s lead to membrane proliferation in Yeasts. May be possible to express an enzymatically inactive P450 that elicits proliferation via membrane anchor. Expression of secreted enzymes, such as invertase (SUC2) can lead to membrane genesis in yeasts.

Growth with compounds that lead to membrane proliferation.

Altering genetics of peroxisome proliferation.

Enzymes that are Candidates for manipulating either by modulating or eliminating expression, or substituting homologues or engineered enzymes, e.g. that eliminate feedback or end product inhibition.

5.6. PRODUCTION OF LONG-CHAIN Ω-HYDROXY FATTY ACIDS

Whole-cell biocatalysts currently used to oxidize long chain fatty acids include Candida tropicalis, Candida cloacae, Cryptococcus neoforman and Corynebacterium sp. One preferred microorganisms is Candida tropicalis ATCC20962 in which the β-oxidation pathway is blocked by disrupting POX 4 and POX 5 genes which respectively encode the acyl-coenzyme A oxidases PXP-4 (SEQ ID NO: 134) and PXP-5 (SEQ ID NO: 135). This prevents metabolism of the fatty acid by the yeast (compare FIGS. 2 and 3). The fatty acids or alkynes used have 14 to 22 carbon atoms, can be natural materials obtained from plants or synthesized from natural fatty acids, such as lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid (ω3, C18:3) ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, 12-OH—C18:1), erucic acid (C22:1), epoxy stearic acid. Examples of other substrates that can be used in biotransformations to produce α,ω-dicarboxylic acid and ω-hydroxyacid compounds are 7-tetradecyne and 8-hexadecyne. Disclosed herein, naturally derived fatty acids, chemically or enzymatically modified fatty acids, n-alkane, n-alkene, n-alkyne and/or fatty alcohols that have a carbon chain length from 12 to 22 are used as carbon sources for the yeast-catalyzed biotransformation. For example, Candida tropicalis ATCC20962 can be used as a catalyst under aerobic conditions in liquid medium to produce ω-hydroxy fatty acids and α,ω-dicarboxylic acids. Candida tropicalis ATCC20962 is initially cultivated in liquid medium containing inorganic salts, nitrogen source and carbon source. The carbon source for initial cultivations can be saccharide such as sucrose, glucose, sorbitol, etc., and other carbohydrates such as glycerol, acetate and ethanol. Then, the substrate such as naturally derived fatty acids, chemically or enzymatically modified fatty acids, n-alkane, n-alkene, n-alkyne and fatty alcohol for oxidation of terminal methyl or hydroxyl moieties is added into the culture. The pH is adjusted to 7.5-8.0 and fermentations are conducted under aerobic conditions with agitation in a shaker incubator, fermentor or other suitable bioreactor.

For example, the fermentation process may be divided into two phases: a growth phase and a transformation phase in which ω-oxidation of the substrate is performed. The seeds inoculated from fresh agar plate or glycerol stock are firstly cultivated in a preculture medium for 16-20 hours, at 30° C. and pH 6.5 in a shaker. Subsequently, this culture is used to inoculate the conversion medium with ω-substrates. The growth phase of the culture is performed for 10-12 hours to generate high cell density cultures at pH 6.5 and 30° C. The transformation phase is begun with addition of the fatty acid or other substrate for the bio-oxidation. The medium pH is adjusted to 7.5-8.0 by addition of a base solution. Co-substrates are fed during the transformation phase to provide energy for cell growth. By use of this method, the terminal methyl group of fatty acids, synthetically derived substrates, n-alkanes, n-alkenes, n-alkynes and/or fatty alcohols that have a carbon chain length from 12 to 22 are converted to a hydroxyl or carboxyl group.

5.7. GENETIC MODIFICATIONS OF CANDIDA TROPICALIS

Yeasts of the genus Candida including Candida tropicalis contains two pathways for the metabolism of fatty acids: ω-oxidation and β-oxidation. These pathways are shown schematically in FIG. 2, together with some classes of enzymes capable of catalyzing the chemical conversions in each pathway. In order for Candida to be used to transform fatty acids into useful compounds-such as diacids and hydroxyl fatty acids, or high energy compounds, or other chemicals it is advantageous to eliminate metabolic pathways that can divert either the substrates or products of the desired pathway. For example it may be desirable to prevent Candida from metabolizing fatty acids through the β-oxidation pathway, so that more fatty acids are available for conversion to α,ω-diacids and ω-hydroxy fatty acids by the ω-oxidation pathway. This can be accomplished by deleting the acyl coenzyme A oxidase genes, as shown in FIG. 2 (Picataggio et al., 1992, Biotechnology (N Y): 10, 894-8.; Picataggio et al., 1991, Mol Cell Biol: 11, 4333-9.).

Candida tropicalis strains lacking both alleles of each of two acyl coenzyme A oxidase isozymes, encoded by the pox4 and pox5 genes, are efficient biocatalysts for the production of α,ω-diacids (Picataggio et al., 1992, Biotechnology (N Y): 10, 894-8; Picataggio et al., 1991, Mol Cell Biol: 11, 4333-4339). However for the production of ω-hydroxy fatty acids, additional enzymes must be eliminated to prevent the oxidation of the ω-hydroxyl group to a carboxyl group.

To prevent the oxidation of hydroxyl groups to carboxyl groups, in some embodiments it is particularly advantageous to eliminate or inactivate one or more genes encoding a cytochrome P450.

To prevent the oxidation of hydroxyl groups to carboxyl groups, in some embodiments it is particularly advantageous to eliminate or inactivate one or more genes encoding a fatty alcohol dehydrogenase.

To prevent the oxidation of hydroxyl groups to carboxyl groups, in some embodiments it is particularly advantageous to eliminate or inactivate one or more genes encoding an alcohol dehydrogenase.

In one embodiment yeast genes can be inactivated by deleting regions from the yeast genome that encode a part of the yeast gene that encodes the protein product (the open reading frame) so that the full-length protein can no longer be made by the cell. In another embodiment yeast genes can be inactivated by inserting additional DNA sequences into the part of the yeast gene that encodes the protein product so that the protein that is made by the cell contains changes that prevent it from functioning correctly. In another embodiment yeast genes are inactivated by inserting or deleting sequences from control regions of the gene, so that the expression of the gene is no longer correctly controlled; for example additions or deletions to the promoter can be used to prevent transcription of the gene, additions or deletions to the polyadenylation signal can be used to affect the stability of the mRNA, additions or deletions to introns or intron splicing signals can be used to prevent correct splicing or nuclear export of the processed mRNA.

For the production of oxidized compounds in yeast-including ω-hydroxy fatty acids and high energy compounds, it may also be advantageous to add certain new genes into the yeast cell. For example to facilitate the production of ω-hydroxy fatty acids from fatty acids with different chain lengths or degrees or positions of unsaturation, the enzymes that are naturally present in the yeast are often inadequate; they may oxidise the fatty acid to the ω-hydroxy fatty acid too slowly, they may only oxidise a subset of the fatty acids in a mixture to their corresponding ω-hydroxy fatty acids, they may oxidise the fatty acid in the wrong position or they may oxidise the ω-hydroxy fatty acid itself to a diacid. Advantageous enzymes could thus be those that oxidise a compound to the corresponding hydroxylated compound more rapidly, those that oxidise a fatty acid to its corresponding ω-hydroxy fatty acid more rapidly, those that accept as substrates a wider range of substrates and those that do not over-oxidise target compounds including ω-hydroxy fatty acids to diacids.

To achieve novel phenotypes in Candida species, including the ability to perform biotransformations such as novel chemical conversions, or increased rates of conversion of one or more substrates to one or more products, or increased specificity of conversion of one or more substrates to one or more products, or increased tolerance of a compound by the yeast, or increased uptake of a compound by the yeast, it may be advantageous to incorporate a gene encoding a polypeptide into the genome of the yeast.

Preferred sites of integration include positions within the genome where the gene would be under control of a promoter that transcribes high levels of an endogenous protein, or under control of a promoter that leads to regulated transcription for example in response to changes in the concentrations of one or more compound in the cellular or extracellular environment. Examples of preferred sites of integration include sites in the genome that are under control of the promoter for an isocitrate lyase gene, sites in the genome that are under control of the promoter for a cytochrome P450 gene, sites in the genome that are under control of the promoter for a fatty alcohol oxidase gene and sites in the genome that are under control of the promoter for an alcohol dehydrogenase gene to obtain high levels of expression of a polypetidepolypeptide or expression of a polypeptide under specific circumstances.

To achieve such novel phenotypes in Candida species, it may be advantageous to modify the activity of a polypeptide by altering its sequence, and to test the effect of the polypeptide with altered sequence within the yeast. Polypeptides of particular interest for conferring the ability to synthesize novel hydroxyfatty acids include cytochrome P450s and their reductases, glycosyl transferases and desaturases. A preferred method for testing the effect of sequence changes in a polypeptide within yeast is to introduce a plurality of genes of known sequence, each encoding a unique modified polypeptide, into the same genomic location in a plurality of strains.

Some embodiments described herein make use of a selective marker. A selective marker can be a gene that produces a selective advantage for the cells under certain conditions such as a gene encoding a product that confers resistance to an antibiotic or other compound that normally inhibits the growth of the host cell.

A selective marker can be a reporter, such as, for example, any nucleic acid sequence encoding a detectable gene product. The gene product may be an untranslated RNA product such as mRNA or antisense RNA. Such untranslated RNA may be detected by techniques known in the art, such as PCR, Northern or Southern blots. The selective marker may encode a polypeptide, such as a protein or peptide. A polypeptide may be detected immunologically or by means of its biological activity. The selective marker may be any known in the art. The selective marker need not be a natural gene. Useful selective markers may be the same as certain natural genes, but may differ from them either in terms of non-coding sequences (for example one or more naturally occurring introns may be absent) or in terms of coding sequences. One example of such a detectable gene product is one that causes the yeast to adopt a unique characteristic color associated with the detectable gene product. For example, if the targeting construct contains a selective marker that is a gene that directs the cell to synthesize a fluorescent protein, then all of the colonies that contain the fluorescent protein are carrying the targeting construct and are therefore likely to be integrants. Thus the cells that will be selected for further analysis are those that contain the fluorescent protein.

The selective marker may encode a protein that allows the yeast cell to be selected by, for example, a nutritional requirement. For example, the selective marker may be the ura4 gene that encodes orotidine-5′-phosphate decarboxylase. The ura4 gene encodes an enzyme involved in the biosynthesis of uracil and offers both positive and negative selection. Only cells expressing ura4 are able to grow in the absence of uracil, where the appropriate yeast strain is used. Cells expressing ura4 die in the presence of 5-fluoro-orotic acid (FOA) as the ura4 gene product converts FOA into a toxic product. Cells not expressing ura4 can be maintained by adding uracil to the medium. The sensitivity of the selection process can be adjusted by using medium containing 6-azauracil, a competitive inhibitor of the ura4 gene product. The his3 gene, which encodes imidazoleglycerol-phosphate dehydratase, is also suitable for use as a selective marker that allows nutritional selection. Only cells expressing his3 are able to grow in the absence of histidine, where the appropriate yeast strain is used.

The selective marker may encode for a protein that allows the yeast to be used in a chromogenic assay. For example, the selective marker may be the lacZ gene from Escherichia coli. This encodes the β-galactosidase enzyme which catalyses the hydrolysis of β-galactoside sugars such as lactose. The enzymatic activity of the enzyme may be assayed with various specialized substrates, for example X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) or o-nitrophenyl-β-D-galactopyranoside, which allow selective marker enzyme activity to be assayed using a spectrophotometer, fluorometer or a luminometer.

In some embodiments, the selective marker comprises a gene that encodes green fluorescent protein (GFP), which is known in the art.

In some embodiments, the selective marker encodes a protein that is capable of inducing the cell, or an extract of a cell, to produce light. For example, the selective marker encodes luciferase in some embodiments. The use of luciferase is known in the art. They are usually derived from firefly (Photinous pyralis) or sea pansy (Renilla reniformis). The luciferase enzyme catalyses a reaction using D-luciferin and ATP in the presence of oxygen and Mg²⁺ resulting in light emission. The luciferase reaction is quantitated using a luminometer that measures light output. The assay may also include coenzyme A in the reaction that provides a longer, sustained light reaction with greater sensitivity. An alternative form of enzyme that allows the production of light and which can serve as a selective marker is aequorin, which is known in the art.

In some embodiments the selective marker encodes β-lactamase. This selective marker has certain advantages over, for example, lacZ. There is no background activity in mammalian cells or yeast cells, it is compact (29 kDa), it functions as a monomer (in comparison with lacZ which is a tetramer), and has good enzyme activity. This may use CCF2/AM, a FRET-based membrane permeable, intracellularly trapped fluorescent substrate. CCF2/AM has a 7-hydroxycoumarin linked to a fluorescein by a cephalosporin core. In the intact molecules, excitation of the coumarin results in efficient FRET to the fluorescein, resulting in green fluorescent cleavage of the CCF2 by β-lactamase results in spatial separation of the two dyes, disrupting FRET and causing cells to change from green to blue when viewed using a fluorescent microscope. The retention of the cleaved product allows the blue colour to develop over time, giving a low detection limit of, for example, 50 enzyme molecules per cell. This results in the selective maker being able to be assayed with high sensitivity. It also allows the ability to confirm results by visual inspection of the cells or the samples.

In some embodiments, the selective marker comprises any of the aforementioned genes under the control of a promoter. In some embodiments, the selective marker comprises any of the aforementioned genes under the control of a promoter as well as one or more additional regulatory elements, such as upstream activating sequences (UAS), termination sequences and/or secretory sequences known in the art. The secretory sequences may be used to ensure that the product of the reporter gene is secreted out of the yeast cell.

5.7.1. Methods for Deletion of Sequences from the Candida Genome

Many yeasts recombine DNA in regions of sequence homology. A linear DNA molecule that is introduced into a yeast cell can recombine homologously with the chromosomal DNA if its ends share sufficient sequence identity with chromosomal sequences. Since the sequences of the ends of the DNA molecule are the primary determinant of where in the yeast chromosome the homologous recombination event occurs, it is possible to construct a DNA molecule that encodes one or more functional genes, and to target that molecule to integrate at a specific location in the yeast chromosome. In this way, yeast genes in the chromosome or mitochondria may be disrupted, by interrupting the gene sequence with other sequences.

In one embodiment, a DNA construct comprises two sequences with homology to two sequences in the target yeast genome (“targeting sequences”), separated by a selective marker, as shown in FIG. 11. The two target sequences within the yeast genome are preferably located on the same molecule of DNA (e.g. the same nuclear or mitochondrial chromosome), and are preferably less than 1,000,000 base pairs apart, more preferably they are less than 100,000 base pairs apart, and more preferably they are less than 10,000 base pairs apart. Cells containing a genomic integration of the targeting construct can be identified using the selective marker.

A schematic representation of one form of a DNA molecule for yeast genomic integration (a “genomic targeting construct”) is shown in FIG. 4. In this embodiment the genomic targeting construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast genome. In some embodiments these sequences are each at least 100 base pairs in length, or between 100 and 300 base pairs in length. The targeting sequences are preferably 100% identical to sequences in the host genome or between 95% and 100% identical to sequences in the host genome. Between these targeting sequences are two sites recognized by a site-specific recombinase such as the natural or modified versions of cre or flp or PhiC31 recombinases or serine recombinases such as those from bacteriophage R4 or bacteriophage TP901-1. Between the two site specific recombinase recognition sites are functional sequence elements which may include sequences that encode a site-specific recombinase that recognizes the recombinase sites and which may also encode a selective marker as illustrated in FIG. 4. In one embodiment this DNA construct incorporates the “SAT1 flipper”, a DNA construct for inserting and deleting sequences into the chromosome of Candida (Reuss et al., 2004, Gene: 341, 119-277). In the “SAT1 flipper” the recombinase is the flp recombinase from Saccharomyces cerevisiae (Vetter et al., 1983, Proc Natl Acad Sci USA: 80, 7284-8) (FLP) and the flanking sequences recognized by the recombinase are recognition sites for the flp recombinase (FRT). The selective marker is the gene encoding resistance to the Nourseothricin resistance marker from transposon Tn1825 (Tietze et al., 1988, J Basic Microbiol: 28, 129-36). The entire construct can then be targeted to the Candida chromosome by adding flanking sequences with homology to a gene in the Candida chromosome. The DNA sequence of the SAT1-flipper is SEQ ID NO: 1.

Yeast preferentially recombines linear DNA. It is therefore advantageous to prepare the targeting construct as a linear molecule prior to transforming it into the yeast target. In some embodiments it is desirable to prepare and propagate the targeting construct as plasmid DNA in a bacterial host such as E. coli. For propagation in a bacterial host it is generally preferred that plasmid DNA be circular. It is thus sometimes necessary to convert the targeting construct from a circular molecule to a linear molecule. Furthermore for propagation of the targeting construct in a bacterial host, additional sequence elements may be necessary, so a targeting construct may, in addition to the elements shown in FIGS. 4 and 7, comprise an origin of replication and a bacterial selectable marker. It may therefore be advantageous to place restriction sites in the targeting construct to cleave between the elements of the targeting construct shown in FIGS. 4 and 7 and the elements not shown but required for propagation in a bacterial host. Cleavage with restriction enzymes that recognize these sites will linearize the DNA and leave the targeting sequences at the ends of the molecule, favoring homologous recombination with the target host genome. One of ordinary skill in the art will recognize that there are alternative ways to obtain linear DNA, for example by amplifying the desired segment of DNA by PCR. It is also possible to prepare the DNA directly and transform it into the target yeast strain without propagating as a plasmid in a bacterial host.

Introduction of the linearized targeting construct into a yeast host cell such as a Candida host cell is followed by homologous recombination catalyzed by host cell enzymes. This event is represented schematically in FIG. 5. Homologous recombination occurs between each of the two targeting sequences in the genomic targeting construct and the homologous sites in the yeast genome. The result is an integration of the targeting construct into the genomic DNA. Cells containing a genomic integration of the targeting construct can be identified using the selective marker.

Cells containing a genomic integration of the targeting construct can optionally be tested to ensure that the integration has occurred at the desired site within the genome. In one embodiment, such testing is performed by amplification of a section of the genomic DNA by the polymerase chain reaction. Integration of the targeting construct into the yeast genome will replace genomic sequences with targeting construct sequences. This replacement may be detected by a difference in size of amplicon using oligonucleotide primers that anneal to sequences outside the targeted sequence. This is illustrated in FIG. 10. One of ordinary skill in the art will readily appreciate that there are many alternative ways to design oligonucleotides to produce diagnostic amplicons using the polymerase chain reaction. For example one oligonucleotide that anneals inside the targeted region and one oligonucleotide that anneals outside but close to the targeted region can be used to produce an amplicon from the natural genomic sequence but will not produce an amplicon if the targeting construct has eliminated the targeted genomic sequence. Conversely one oligonucleotide that anneals inside the targeting construct and one oligonucleotide that anneals outside but close to the targeted region outside will not produce an amplicon from the natural genomic sequence but will produce an amplicon if the targeting construct has integrated at the targeted genomic location. In general oligonucleotide pairs for producing diagnostic amplicons should be oriented with their 3′ ends towards each other and the sites in the genome where the two oligonucleotides anneal should be separated by between 100 and 10,000 bases, more preferably by between 150 and 5,000 bases and more preferably by between 200 and 2,000 bases. In some instances it may not be possible to distinguish between two possible genotypes based on the size of the amplicons produced by PCR from genomic DNA. In these cases an additional test is possible, for example digestion of the amplicon with one or more restriction enzymes and analysis of the sizes may enable the two possible genotypes to be distinguished, or sequencing of the amplicon may enable the two possible genotypes to be distinguished.

The same selectable marker may be used for the disruption of more than one genomic target. This can be achieved by removing the selectable marker from the yeast genome after each disruption. In one embodiment, this is achieved when the selectable marker separates two sites that are recognized by a recombinase. When the recombinase is present and active, it effects a recombination reaction between the two sites, excising the sequences between them. In the targeting construct shown in FIG. 6 this is done by induction of the gene encoding the recombinase present in the targeting construct. Expression of the recombinase causes a recombination event between the two recombinase recognition sites of the targeting construct, as shown schematically in FIG. 6. The result is that the sequences between the two recombinase sites are excised from the genome. In other embodiments it is possible to integrate a recombinase into a second site in the host genome instead of having it present in the targeting construct.

Cells from which a genomic integration of the targeting construct has been excised can optionally be tested to ensure that the excision has occurred by testing cells from individual colonies to determine whether they still carry the selective marker. In some embodiments, such testing is performed by amplification of a section of the genomic DNA by the polymerase chain reaction. Excision of part of the targeting construct from the yeast genome may be detected by a difference in size of amplicon using oligonucleotide primers that anneal to sequences outside the targeted sequence. This is illustrated in FIG. 10. One of ordinary skill in the art will readily appreciate that there are many alternative ways to design oligonucleotides to produce diagnostic amplicons using the polymerase chain reaction. For example one oligonucleotide that anneals inside the targeting construct (example. g. within the selective marker) and one oligonucleotide that anneals outside but close to the targeted region can be used to produce an amplicon from the integrated targeting construct but will not produce an amplicon if the targeting construct has been excised. In general oligonucleotide pairs for producing diagnostic amplicons should be oriented with their 3′ ends towards each other and the sites in the genome where the two oligonucleotides anneal should be separated by between 100 and 10,000 bases, more preferably by between 150 and 5,000 bases and more preferably by between 200 and 2,000 bases. In some instances it may not be possible to distinguish between two possible genotypes based on the size of the amplicons produced by PCR from genomic DNA. In these cases an additional test is possible, for example digestion of the amplicon with one or more restriction enzymes and analysis of the sizes may enable the two possible genotypes to be distinguished, or sequencing of the amplicon may enable the two possible genotypes to be distinguished.

In some embodiments it may be advantageous to delete sequences whose deletion will result in the inactivation of a cytochrome P450; in some embodiments it may be advantageous to delete sequences whose deletion will result in the inactivation of a fatty alcohol oxidase; in some embodiments it may be advantageous to delete sequences whose deletion will result in the inactivation of an alcohol dehydrogenase.

5.7.2. Methods for Addition of Sequences to the Candida Genome

In some embodiments, new DNA sequences can be inserted into the yeast genome at a specific location using variations of the targeting construct. Because many yeasts recombine DNA in regions of sequence homology, a linear DNA molecule that is introduced into a yeast cell can recombine homologously with the chromosomal DNA if its ends share sufficient sequence identity with chromosomal sequences. It is thus possible to insert a DNA sequence into the yeast genome at a specific location by flanking that sequence with sequences homologous to sequences within the yeast genome that surround the desired genomic insertion site. Such replacements are quite rare, generally occurring less than 1 time in 1,000 yeast cells, so it is often advantageous to use a selective marker to indicate when new DNA sequences have been incorporated into the yeast genome. A selective marker can be used in conjunction with a sequence to be integrated into the yeast genome by modifying the strategy described for deleting sequences form the yeast genome.

If a targeting construct comprises additional sequences between one of the targeting sequences and the proximal recombinase site, those sequences will be retained in the genome following integration and excision of the targeting construct. An example of such a construct is shown in FIG. 7, with the additional sequences indicated as “insertion sequences.” Integration of the targeting construct for insertion into the yeast genome is shown schematically in FIG. 8. Homologous recombination occurs between each of the two targeting sequences in the genomic targeting construct and the homologous sites in the yeast genome. The result is an integration of the targeting construct into the genomic DNA. Cells containing a genomic integration of the targeting construct can be identified using the selective marker.

Cells containing a genomic integration of the targeting construct can optionally be tested to ensure that the integration has occurred at the desired site within the genome. In one embodiment, such testing may be performed by amplification of a section of the genomic DNA by the polymerase chain reaction, for example as illustrated in FIG. 10. One of ordinary skill in the art will readily appreciate that there are many alternative ways to design oligonucleotides to produce diagnostic amplicons using the polymerase chain reaction.

The selectable marker and other sequences from the targeting construct can be removed from the yeast genome using a recombinase-based strategy: the recombinase effects a recombination reaction between the two recombinase sites, excising the sequences between them. In the targeting construct shown in FIG. 7 this is done by induction of the gene encoding the recombinase present in the targeting construct. Expression of the recombinase causes a recombination event between the two recombinase recognition sites of the targeting construct, as shown schematically in FIG. 9. The result is that the sequences between the two recombinase sites are excised from the genome, leaving the insertion sequences integrated into the yeast genome.

Cells to which a genomic integration has been introduced can optionally be tested to ensure that the addition has occurred correctly by polymerase chain reaction amplification of DNA from the yeast genome. These amplicons may then be tested to measure their size (for example by agarose gel electrophoresis), or their sequence may be determined to ensure that precisely the desired changes have been effected.

In some embodiments, it may be advantageous to insert sequences into a site in the genome that is known to be transcriptionally active. For example inserting a sequence encoding a polypeptide into a genomic site where transcription is regulated by a promoter that expresses high levels of mRNA can produce high levels of mRNA encoding the polypeptide. In some embodiments this can be done by replacing a polypeptide encoding sequence in the genome with a sequence encoding a different polypeptide, for example using the genomic targeting constructs of the form shown in FIG. 7.

In some embodiments, the insertion of a sequence encoding a polypeptide into a genomic site where transcription is regulated by a promoter that expresses high levels of mRNA is accomplished by adding a polypeptide encoding sequence into the genome at a position where a part of the genomic sequence is duplicated so that the gene that was originally present in the genome remains. In some embodiments this can be effected using a DNA construct comprising a promoter sequence found in the yeast genome positioned such that transcription initiated by the promoter produces RNA that can subsequently encode the polypeptide. Such a construct also comprises a selectable marker that will function in the yeast and optionally a selectable marker that will function in a bacterial host. These may optionally be the same selectable marker. An example of such a construct is shown in FIG. 21. Integration of this construct into the yeast genome is shown schematically in FIG. 22.

In some embodiments, a sequence encoding a polypeptide is inserted under control of the promoter for an isocitrate lyase gene or the promoter for a cytochrome P450 gene including the promoter of CYP52A12 or the promoter of CYP52A13 or the promoter of CYP52A14 or the promoter of CYP52A17 or the promoter of CYP52A18 or the promoter for a fatty alcohol oxidase gene including the promoter of FAO1 or the promoter of FAO1B or the promoter of FAO2A or the promoter of FAO2B, or the promoter for an alcohol dehydrogenase gene including the promoter of ADH-A4 or the promoter of ADH-A4B or the promoter of ADH-B4 or the promoter of ADH-B4B or the promoter of ADH-A10 or the promoter of ADH-B11 or the promoter of ADH-A10B or the promoter of ADH-B11B to obtain high levels of expression of a polypetide.

In addition to naturally occurring enzymes, modified enzymes may be added into the host genome. For example enzymes may be altered by incorporating systematically varied sets of amino acid changes, with the resulting changes in phenotypes measured and used to identify sequence changes conferring improved function. See, for example, United States Patent Publications Nos. 20060136184 and 20080050357; Liao et al., 2007, BMC Biotechnol 7, 16; Ehren et al., 2008, Protein Eng Des Sel 21, 699-707 and Heinzelman et al., 2009, Proc Natl Acad Sci USA 106, 5610-5615. Using these methods, modified versions of enzymes may be obtained that confer on the host cell an improved ability to utilize one or more substrate or an improved ability to perform one or more chemical conversion. A gene that has been modified by these methods may be made more useful in the genome of the host by amplification, that is by genetic manipulations causing the presence of more than one copy of the gene within the host cell genome and frequently resulting in higher activity of the gene.

5.7.3. Other Microorganisms of Interest for the Production of Oxidized Fatty Acids

Homology-based recombination occurs in the Saccharomycetacaeae Family (which is in the Saccharomycotina Subphylum); Saccharomycetacaeae include the Genera Ascobotryozyma, Candida, Citeromyces, Debaryomyces, Dekkera (Brettanomyces), Eremothecium, Issatchenkia, Kazachstania, Kluyveromyces, Kodamaea, Kregervanrija, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia (Hansenula), Saccharomyces, Saturnispora, Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Zygosaccharomyces. The deletion and insertion methods described here are therefore likely to work in these Genera.

Within the Subphylum Saccharomycotina is a monophyletic clade containing organisms that translate CTG as serine instead of leucine (Fitzpatrick et al., A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis BMC Evolutionary Biology 2006, 6:99) including the species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. Of particular interest are modifications of the activities of cytochrome P450s, fatty alcohol oxidases and alcohol dehydrogenases to modulate the host's production of oxidized molecules by yeasts in this clade. Yeast species of particular interest and industrial relevance within this clade include Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bitumimphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida fioricola, Candida fioris, Candida fiosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida maris, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, Candida zeylanoides

5.7.4. Engineering of Additional Enzymes into Candida to Further Diversify Structures of Products Formed

Different fatty acids are hydroxylated at different rates by different cytochrome P450s. To achieve efficient hydroxylation of a desired fatty acid feedstock, one strategy is to express P450 enzymes within Candida that are active for ω-hydroxylation of a wide range of highly abundant fatty acid feedstocks. Of particular interest are P450 enzymes that catalyze ω-hydroxylation of lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and α-linolenic acid (ω3, C18:3). Examples of P450 enzymes with known ω-hydroxylation activity on different fatty acids that may be cloned into Candida are the following: CYP94A1 from Vicia sativa (Tijet et al., 1988, Biochemistry Journal 332, 583-589); CYP 94A5 from Nicotiana tabacum (Le Bouquin et al., 2001, Eur J Biochem 268, 3083-3090); CYP78A1 from Zea mays (Larkin, 1994, Plant Mol Biol 25, 343-353); CYP 86A1 (Benveniste et al., 1998, Biochem Biophys Res Commun 243, 688-693) and CYP86A8 (Wellesen et al., 2001, Proc Natl Acad Sci USA 98, 9694-9699) from Arabidopsis thaliana; CYP 92B1 from Petunia hybrida (Petkova-Andonova et al., 2002, Biosci Biotechnol Biochem 66, 1819-1828); CYP102A1 (BM-3) mutant F87 from Bacillus megaterium (Oliver et al., 1997, Biochemistry 36, 1567-1572); and CYP 4 family from mammal and insect (Hardwick, 2008, Biochem Pharmacol 75, 2263-2275).

A second strategy to obtain efficient hydroxylation (or further oxidation of the hydroxy group to an aldehyde or dicarboxylic acid) of a modified fatty acid is to perform the hydroxylation first and then to expose the hydroxylated fatty acid or aldehyde or dicarboxylic acid to an additional enzyme.

For example incorporating one or more desaturase enzymes into engineered Candida would allow the introduction of double bonds into ω-hydroxyl fatty acids or aldehydes or dicarboxylic acids at desired positions. Examples of desaturases with known specificity that may be cloned into Candida are the following: Δ⁴ desaturase from rat liver microsomes (Savile et al., 2001, J Am Chem Soc 123, 4382-4385), Δ⁵ desaturase from Bacillus subtilis (Fauconnot and Buist, 2001, Bioorg Med Chem Lett 11, 2879-2881), Δ⁶ desaturase from Tetrahymena thermophila (Fauconnot and Buist, 2001, J Org Chem 66, 1210-1215), Δ⁹ desaturase from Saccharomyces cerevisiae (Buist and Behrouzian, 1996, J Am Chem Soc 118, 6295-6296); Δ¹¹ desaturase from Spodoptera littoralis (Pinilla et al., 1999, Biochemistry 38, 15272-15277), Δ¹² desaturase from Arabidopsis thaliana (Buist and Behrouzian, 1998, J Am Chem Soc 120, 871-876); Δ¹⁵ desaturase from Caenorhabditis elegans (Meesapyodsuk et al., 2000, Biochemistry 39, 11948-11954). Many other desaturases are known in the literature that can also be expressed in engineered Candida strains including Candida tropicalis strains to introduce unsaturation at specific sites of fatty acid substrates prior to ω-hydroxylation or to catalyze carbon-carbon double bond formation after ω-hydroxylation of fatty acids.

Expression in engineered Candida strains of P450 enzymes that are known in the literature to introduce additional internal hydroxylation at specific sites of fatty acids or ω-hydroxyfatty acids can be used to produce internally oxidized fatty acids or ω-hydroxyfatty acids or aldehydes or dicarboxylic acids. Examples of P450 enzymes with known in-chain hydroxylation activity on different fatty acids that may be cloned into Candida are the following: CYP81B1 from Helianthus tuberosus with ω-1 to ω-5 hydroxylation (Cabello-Hurtado et al, 1998, J Biol Chem 273, 7260-7267); CYP790C1 from Helianthus tuberosus with ω-1 and ω-2 hydroxylation (Kandel et al., 2005, J Biol Chem 280, 35881-35889); CYP726A1 from Euphorbia lagscae with epoxidation on fatty acid unsaturation (Cahoon et al., 2002, Plant Physiol 128, 615-624); CYP152B1 from Sphingomonas paucimobilis with α-hydroxylation (Matsunaga et al., 2000, Biomed Life Sci 35, 365-371); CYP2E1 and 4A1 from human liver with ω-1 hydroxylation (Adas et al., 1999, J Lip Res 40, 1990-1997); P450_(BSβ) from Bacillus substilis with α- and β-hydroxylation (Lee et al., 2003, J Biol Chem 278, 9761-9767); and CYP102A1 (BM-3) from Bacillus megaterium with ω-1, ω-2 and ω-3 hydroxylation (Shirane et al., 1993, Biochemistry 32, 13732-13741).

In addition to naturally occurring enzymes, modified enzymes may be added into the host genome. For example enzymes may be altered by incorporating systematically varied sets of amino acid changes, with the resulting changes in phenotypes measured and used to identify sequence changes conferring improved function. See, for example, United States Patent Publications Nos. 20060136184 and 20080050357; Liao et al., 2007, BMC Biotechnol 7, 16; Ehren et al., 2008, Protein Eng Des Sel 21, 699-707 and Heinzelman et al., 2009, Proc Natl Acad Sci USA 106, 5610-5615. Using these methods, modified versions of cytochrome P450s may be obtained with improved ability to oxidise fatty acids of different lengths (for example C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24) or different degrees of saturation (for example fatty acids with one carbon-carbon double bond, fatty acids with two carbon-carbon double bonds and fatty acids with three carbon-carbon double bonds) or with unsaturated fatty acids where the unsaturated bond is at different positions relative to the carboxyl group and the ω-position, to hydroxy fatty acids or to dicarboxylic fatty acids. Further, using these methods modified versions of fatty alcohol oxidases or alcohol dehydrogenases may be obtained with improved ability to oxidise hydroxy-fatty acids of different lengths (for example C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24) or different degrees of saturation (for example fatty acids with one carbon-carbon double bond, fatty acids with two carbon-carbon double bonds and fatty acids with three carbon-carbon double bonds) or with unsaturated fatty acids where the unsaturated bond is at different positions relative to the carboxyl group and the ω-position. A gene that has been modified by these methods may be made more useful in the genome of the host by amplification, that is by genetic manipulations causing the presence of more than one copy of the gene within the host cell genome and frequently resulting in higher activity of the gene. Expression of one or more additional enzymes may also be used to functionalize the oxidized fatty acid, either the hydroxyl group or more highly oxidized groups such as aldehydes or carboxylic acids

6. BIOTRANSFORMATION EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a complete description of how to practice, make and use exemplary embodiments of the disclosed methods, and are not intended to limit the scope of what is regarded as the invention.

6.1. General Biotransformation Procedure in Shake-Flask

C. tropicalis ATCC20962 from fresh agar plate or glycerol stock was precultured in 30 ml YPD medium consisting of (g1⁻¹): yeast extract, 10; peptone, 10; glucose, 20 and shaken at 250 rpm, 30° for 20 hours in 500 ml flask. After 16 hours of cultivation at 250 rpm, 30° C., preculture was inoculated at 10% (v/v) to 30 ml conversion medium consisting of (g1⁻¹): peptone, 3; yeast extract, 6; yeast nitrogen base, 6.7; acetic acid, 3; K₂HPO₄, 7.2; KH₂PO₄ 9.3; glucose/glycerol, 20 in 500 ml flask and shaked at 250 rpm. The initial concentration of substrate was about 10-20 g l⁻¹. pH was adjusted to 7.5 by addition of 2 mol 1-1 NaOH solution after 12 hour culture. During biotransformation, concentrated ω-substrate (glucose/glycerol/sodium acetate/ethanol) was fed (1-2.5% per day) and pH was maintained at 7.5˜8.0 by addition of NaOH solution. Samples were taken on a daily basis to determine levels of product by LC-MS.

6.2. General Biotransformation Procedure in Fermentor

Fermentation was carried out in 3-1 Bioflo3000 fermentor (New Brunswick Scientific Co., USA) in fed-batch culture. The conversion medium mentioned above was used except for addition of 0.05% antifoam 204 (Sigma) and 0.5% substrate. The seed culture from fresh agar plate or glycerol stock was prepared in 50 ml of conversion medium for 20 hours at 30° C., 250 rpm prior to inoculation into the fermentor vessel. Following inoculation, the culture was maintained at pH 6.3 and grown at 30°, 900 rpm with aeration rate of 1.5 vvm. After 12 hour fermentations (growth phase), biotransformation phase was started with feeding of substrate (2 ml 1⁻¹). Concentrated glucose (500 g l⁻¹) as ω-substrate was fed continuously at the rate of 1.2 g l-1 h-1. During the biotransformation phase, pH was maintained at 7.6 automatically by addition of 4 mol 1⁻¹ NaOH solution. Antifoam (Antifoam 204) was also added to the fermentor as necessary. Samples were taken on a daily basis to determine levels of product by LC-MS.

6.3. General Extraction and Purification Procedure of Biotransformation Products

The fermentation broth was acidified to pH 1.0 with HCl and extracted twice with diethyl ether. To avoid the epoxy ring-opening during acidification, the fermentation broth with products containing epoxy groups was slowly acidified to pH 3.0 with 5 N HCl. Solvent was evaporated under vacuum with a rotary evaporator. The residual obtained was separated by silica gel column chromatography using silica gel 60. The fractions containing impurities, un-reacted mono fatty acids and products were gradually eluted with a mixture of n-hexane/diethyl ether that their ratio ranges from 90:30 to 10:90. The fractions containing same compound were collected together and the solvents were evaporated under vacuum with a rotary evaporator.

7. GENETIC MODIFICATION EXAMPLES

The following examples are set forth so as to provide those of ordinary skill in the art with a description of how to practice, make and use various disclosed exemplary embodiments, and are not intended to limit the scope of what is regarded as the invention.

The strains shown in Table 2 and further described in this section were constructed by the synthesis and cloning of DNA and its subsequent transformation into the appropriate C. tropicalis strain. Table 2 summarizes the DNA sequences synthesized and used in these examples. Table 3 summarizes the C. tropicalis strains constructed in these examples. Section 7.1 describes the methods used for transformation of Candida tropicalis.

TABLE 2 SEQ ID SOURCE/ NAME NO: GI No. CONSTRUCTION APPLICATION SAT1 Flipper 1 50059745 Joachim Morschhauser Source of the SAT1 Flipper CYP52A17 2 29469874 Used to design CYP52A17_Δ CYP52A17_Δ 3 Not Gene synthesis Used to construct applicable CYP52A17::SAT1 CYP52A17::SAT1 4 Not Subcloning of SAT1 Used to delete CYP52A17 applicable flipper into CYP52A17_Δ CYP52A13 5 29469864 Used to design CYP52A13_Δ CYP52A13_Δ 6 Not Gene synthesis Used to construct applicable CYP52A13::SAT1 CYP52A13::SAT1 7 Not Subcloning of SAT1 Used to delete CYP52A13 applicable flipper into CYP52A13_Δ CYP52A18 8 29469876 Used to design CYP52A18_Δ CYP52A18_Δ 9 Not Gene synthesis Used to construct applicable CYP52A18::SAT1 CYP52A18::SAT1 11 Not Subcloning of SAT1 Used to delete CYP52A18 applicable flipper into CYP52A18_Δ CYP52A14 13 29469866 Used to design CYP52A14_Δ_Gene#1179 1179 CYP52A14_Δ 14 Not Gene synthesis Used to construct applicable CYP52A14::SAT1 CYP52A14::SAT1 15 Not Subcloning of SAT1 Used to delete CYP52A14 applicable flipper into CYP52A14_Δ FAO1 16 44194456 Used to design FAO1_Δ FAO1_Δ 17 Not Gene synthesis Used to construct applicable FAO1::SAT1 FAO1::SAT1 18 Not Subcloning of SAT1 Used to delete FAO1 applicable flipper into FAO1_Δ FAO1B 19 Not Used to design FAO1B_Δ applicable FAO1B_Δ 20 Not Assembly PCR. Product Used to construct applicable not cloned. FAO1B::SAT1 FAO1B::SAT1 21 Not Ligation of SAT1 flipper Used to delete FAO1B applicable to assembly PCR product of FAO1B_Δ FAO2A 22 44194479 Used to design FAO2A_Δ FAO2A_Δ 23 Not Gene synthesis Used to construct applicable FAO2A::SAT1 FAO2A::SAT1 24 Not Subcloning of SAT1 Used to delete FAO2A applicable flipper into FAO2A_Δ FAO2B 25 44194514 Used to design FAO2B_Δ FAO2B_Δ 26 Not Gene synthesis Used to construct applicable FAO2B::SAT1 FAO2B::SAT1 27 Not Subcloning of SAT1 Used to delete FAO2B applicable flipper into FAO2B_Δ CYP52A12 28 29469862 Used to design CYP52A12_Δ CYP52A12_Δ 29 Not Gene synthesis Used to construct applicable CYP52A12::SAT1 CYP52A12::SAT1 30 Not Subcloning of SAT1 Used to delete CYP52A12 applicable flipper into CYP52A12_Δ CYP52A12B Not Used to design applicable CYP52A12B_Δ CYP52A12B_Δ 31 Not Gene synthesis Used to construct applicable CYP52A12B::SAT1 CYP52A12B::SAT1 32 Not Subcloning of SAT1 Used to delete CYP52A12B applicable flipper into CYP52A12B_Δ ADH-A4 39 Not Used to design ADH-A4_Δ applicable ADH-A4_Δ 44 Not Gene synthesis Used to construct ADH- applicable A4::SAT1 ADH-A4::SAT1 45 Not Subcloning of SAT1 Used to delete ADH-A4 applicable flipper into ADH-A4_Δ ADH-A4B Not Used to design ADH- applicable A4B_Δ ADH-A4B_Δ 46 Not Gene synthesis Used to construct ADH- applicable A4B::SAT1 ADH-A4B::SAT1 47 Not Subcloning of SAT1 Used to delete ADH-A4B applicable flipper into ADH-A4B_Δ ADH-B4 42 Not Used to design ADH-B4_Δ applicable ADH-B4_Δ 48 Not Gene synthesis Used to construct ADH- applicable B4::SAT1 ADH-B4::SAT1 49 Not Subcloning of SAT1 Used to delete ADH-B4 applicable flipper into ADH-B4_Δ ADH-B4B Not Used to design ADH- applicable B4B_Δ ADH-B4B_Δ 50 Not Gene synthesis Used to construct ADH- applicable B4B::SAT1 ADH-B4B::SAT1 51 Not Subcloning of SAT1 Used to delete ADH-B4B applicable flipper into ADH-B4B_Δ ADH-A10 40 Not Used to design ADH- applicable A10_Δ ADH-A10_Δ 52 Not Gene synthesis Used to construct ADH- applicable A10::SAT1 ADH-A10::SAT1 53 Not Subcloning of SAT1 Used to delete ADH-A10 applicable flipper into ADH-A10_Δ ADH-B11 43 Not Used to design ADH- applicable B11_Δ ADH-B11_Δ 54 Not Gene synthesis Used to construct ADH- applicable B11::SAT1 ADH-B11::SAT1 55 Not Subcloning of SAT1 Used to delete ADH-B11 applicable flipper into ADH-B11_Δ ADH-A10B 56 Not Used to design ADH- applicable A10B_Δ ADH-A10B_Δ 57 Not Gene synthesis Used to construct ADH- applicable A10B::SAT1 ADH-A10B::SAT1 58 Not Subcloning of SAT1 Used to delete ADH-A10B applicable flipper into ADH- A10B_Δ ADH-B11B 59 Not Used to design ADH- applicable B11B_Δ ADH-B11B_Δ 60 Not Gene synthesis Used to construct ADH- applicable B11B::SAT1 ADH-B11B::SAT1 61 Not Subcloning of SAT1 Used to delete ADH-B11B applicable flipper into ADH- B11B_Δ ICL promoter 62 Not Gene synthesis Used as a component of applicable genomic integration and expression constructs (e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) ICL terminator 63 Not Gene synthesis Used as a component of applicable genomic integration and expression constructs(e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) TEF1 promoter 64 Not Gene synthesis Used as a component of applicable genomic integration and expression constructs (e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) EM7 promoter 65 Not Gene synthesis Used as a component of applicable genomic integration and expression constructs (e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) ZeoR 66 Not Gene synthesis of gene Used as a component of applicable optimized for Candida genomic integration and expression constructs (e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) CYC1 transcription 67 Not Gene synthesis Used as a component of terminator applicable genomic integration and expression constructs (e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) pUC origin of 68 Not Gene synthesis Used as a component of replication applicable genomic integration and expression constructs (e.g. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, etc.) CYP52A17 69 Not Gene synthesis Cloned into genomic applicable integration and expression constructs to express (e.g. SEQ ID No: 70) pXICL::CYP52A17 70 Not CYP52A17 cloned into Used to express CYP52A17 applicable genomic integration in Candida tropicalis under vector control of the isocitrate lyase promoter CYP52A13 71 Not Gene synthesis Cloned into genomic applicable integration and expression constructs to express(e.g. SEQ ID NO: 71) pXICL::CYP52A13 72 Not CYP52A13 cloned into Used to express CYP52A13 applicable genomic integration in Candida tropicalis under vector control of the isocitrate lyase promoter CYP52A12 73 Not Gene synthesis Cloned into genomic applicable integration and expression constructs to express(e.g. SEQ ID NO: 74) pXICL::CYP52A12 74 Not CYP52A12 cloned into Used to express CYP52A12 applicable genomic integration in Candida tropicalis under vector control of the isocitrate lyase promoter promoter mCherry 75 Not Gene synthesis Cloned into genomic applicable integration and expression constructs to express mCherry (e.g. SEQ ID NO: 76) pXICL::mCherry 76 Not mCherry cloned into Used to express mCherry in applicable genomic integration Candida tropicalis under vector control of the isocitrate lyase promoter

TABLE 3 Strain Name Genotype Description DP1 ura3A/ura3B American Type Culture Collection (ATCC pox5::ura3A/pox5::ura3A 20962) pox4A::ura3A/pox4B::URA3A DP65 DP1 CYP52A17::SAT1 Electroporation of DP1 with CYP52A17::SAT1 (SEQ ID NO: 4) and selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into CYP52A17 DP78 DP1 ΔCYP52A17 Growth of DP65 with maltose followed by agar plate screen for loss of nourseothricin resistance and PCR screen for excision of targeting construct from CYP52A17 DP107 DP1 ΔCYP52A17 Electroporation of DP78 with CYP52A13::SAT1 CYP52A13::SAT1 (SEQ ID NO: 7) and selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into CYP52A13 DP113 DP1 ΔCYP52A17 ΔCYPS2A13 Growth of DP107 with maltose followed by agar plate screen for loss of nourseothricin resistance and PCR screen for excision of targeting construct from CYP52A13 DP140 DP1 Electroporation of DP113 with ΔCYP52A17/CYP52A18::SAT1 CYP52A18::SAT1 (SEQ ID NO: 11) and ΔCYP52A13 selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into CYP52A18 DP142 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP140 with maltose followed ΔCYP52A13 by agar plate screen for loss of nourseothricin resistance and PCR screen for excision of targeting construct from CYP52A18 DP170 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP142 with ΔCYP52A13/CYP52A14::SAT1 CYP52A14::SAT1(SEQ ID NO: 15) and selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into CYP52A14 DP174 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP170 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of nourseothricin resistance and PCR screen for excision of targeting construct from CYP52A14 DP182 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP174 with ΔCYP52A13/ΔCYP52A14 FAO1::SAT1(SEQ ID NO: 18) and FAO1::SAT1 selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into FAO1 DP186 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP182 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1 nourseothricin resistance and PCR screen for excision of targeting construct from FAO1 DP197 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP186 with ΔCYP52A13/ΔCYP52A14 pXICL::mCherry (SEQ ID NO: 76) and ΔFAO1 pXICL::mCherry selection for zeocin resistance followed by PCR screens for targeting construct insertion into the isocitrate lyase gene DP201 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP186 with ΔCYP52A13/ΔCYP52A14 pXICL::CYP52A17 (SEQ ID NO: 70) and ΔFAO1 pXICL::CYP52A17 selection for zeocin resistance followed by PCR screens for targeting construct insertion into the isocitrate lyase gene DP238 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP186 with ΔCYP52A13/ΔCYP52A14 FAO1B::SAT1(SEQ ID NO: 21) and ΔFAO1/FAO1B::SAT1 selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into FAO1B DP240 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP238 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen for excision of targeting construct from FAO1B DP255 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP240 with ΔCYP52A13/ΔCYP52A14 FAO2A::SAT1(SEQ ID NO: 21) and ΔFAO1/ΔFAO1B FAO2A::SAT1 selection for nourseothricin resistance followed by PCR screens for targeting construct insertion into FAO2A DP256 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP255 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B ΔFAO2A nourseothricin resistance and PCR screen for excision of targeting construct from FAO2A DP258 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP256 with DP259 ΔCYP52A13/ΔCYP52A14 FAO2B::SAT1(SEQ ID NO: 27) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/FAO2B::SAT1 followed by PCR screens for targeting construct insertion into FAO2B DP261 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP259 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from FAO2B DP268 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP261 with ΔCYP52A13/ΔCYP52A14 CYP52A12::SAT1 (SEQ ID NO: 30) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/ΔFAO2B followed by PCR screens for targeting CYP52A12::SAT1 construct insertion into CYP52A12 DP272 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP268 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B ΔCYP52A12 for excision of targeting construct from CYP52A12 DP282 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP272 with ΔCYP52A13/ΔCYP52A14 CYP52A12B::SAT1 (SEQ ID NO: 32) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/ΔFAO2B followed by PCR screens for targeting ΔCYP52A12/CYP52A12B::SAT1 construct insertion into CYP52A12B DP283 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP282 with maltose followed DP284 ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B CYP52A12B DP387 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP283 with ADH- ΔCYP52A13/ΔCYP52A14 A4::SAT1(SEQ ID NO: 45) and selection ΔFAO1/ΔFAO1B for nourseothricin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into ADH-A4 ADH-A4::SAT1 DP388 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP387 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-A4 ΔADH-A4 DP389 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP388 with ADH- ΔCYP52A13/ΔCYP52A14 A4B::SAT1 (SEQ ID NO: 47) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/ΔFAO2B followed by PCR screens for targeting ΔCYP52A12/ΔCYP52A12B construct insertion into ADH-A4B ΔADH-A4/ADH-A4B::SAT1 DP390 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP389 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-A4B ΔADH-A4/ΔADH-A4B DP397 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP390 with ADH- ΔCYP52A13/ΔCYP52A14 B4::SAT1 (SEQ ID NO: 49) and selection ΔFAO1/ΔFAO1B for nourseothricin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into ADH-B4 ΔADH-A4/ΔADH-A4B ADH- B4::SAT1 DP398 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP397 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-B4 ΔADH-A4/ΔADH-A4B ΔADH- B4 DP409 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP398 with ADH- ΔCYP52A13/ΔCYP52A14 B4B::SAT1 (SEQ ID NO: 49) and selection ΔFAO1/ΔFAO1B for nourseothricin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into ADH-B4B ΔADH-A4/ΔADH-A4B ΔADH- B4/ADH-B4B::SAT1 DP411 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP409 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-B4B ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B DP415 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP411 with ADH- ΔCYP52A13/ΔCYP52A14 A10::SAT1 (SEQ ID NO: 53) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/ΔFAO2B followed by PCR screens for targeting ΔCYP52A12/ΔCYP52A12B construct insertion into ADH-A10 ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ADH-A10::SAT1 DP416 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP415 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-A10 ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH-A10 DP417 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP416 with ADH- ΔCYP52A13/ΔCYP52A14 B11::SAT1 (SEQ ID NO: 55) and selection ΔFAO1/ΔFAO1B for nourseothricin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into ADH-B11 ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH-A10 ADH-B11::SAT1 DP421 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP417 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss ΔFAO1/ΔFAO1B of nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-B11 ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH-A10 ΔADH-B11 DP423 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP421 with ADH- DP424 ΔCYP52A13/ΔCYP52A14 A10B::SAT1 (SEQ ID NO: 58) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/ΔFAO2B followed by PCR screens for targeting ΔCYP52A12/ΔCYP52A12B construct insertion into ADH-A10B ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH- A10/ADH-A10B::SAT1 ΔADH- B11 DP427 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP421 with DP428 ΔCYP52A13/ΔCYP52A14 pXICL::CYP52A17 (SEQ ID NO: 70) and ΔFAO1/ΔFAO1B selection for zeocin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into the isocitrate lyase gene ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH-A10 ΔADH-B11 pXICL::CYP52A17 DP431 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP424 with maltose followed ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-A10B ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH- A10/ΔADH-A10B ΔADH-B11 DP433 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP431 with ADH- DP434 ΔCYP52A13/ΔCYP52A14 B11B::SAT1 (SEQ ID NO: 61) and ΔFAO1/ΔFAO1B selection for nourseothricin resistance ΔFAO2A/ΔFAO2B followed by PCR screens for targeting ΔCYP52A12/ΔCYP52A12B construct insertion into ADH-B11B ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH- A10/ΔADH-A10B ΔADH- B11/ADHB11B::SAT1 DP436 DP1 ΔCYP52A17/ΔCYP52A18 Growth of DP433 with maltose followed DP437 ΔCYP52A13/ΔCYP52A14 by agar plate screen for loss of ΔFAO1/ΔFAO1B nourseothricin resistance and PCR screen ΔFAO2A/ΔFAO2B for excision of targeting construct from ΔCYP52A12/ΔCYP52A12B ADH-B11B ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH- A10/ΔADH-A10B ΔADH- B11/ΔADHB11B DP522 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP421 with DP523 ΔCYP52A13/ΔCYP52A14 pXICL::CYP52A13 (SEQ ID NO: 72) and ΔFAO1/ΔFAO1B selection for zeocin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into the isocitrate lyase gene ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH-A10 ΔADH-B11 pXICL::CYP52A13 DP526 DP1 ΔCYP52A17/ΔCYP52A18 Electroporation of DP421 with DP527 ΔCYP52A13/ΔCYP52A14 pXICL::CYP52A12 (SEQ ID NO: 74) and ΔFAO1/ΔFAO1B selection for zeocin resistance followed by ΔFAO2A/ΔFAO2B PCR screens for targeting construct ΔCYP52A12/ΔCYP52A12B insertion into the isocitrate lyase gene ΔADH-A4/ΔADH-A4B ΔADH- B4/ΔADH-B4B ΔADH-A10 ΔADH-B11 pXICL::CYP52A12

7.1. General Protocols for Transformation of Candida

The protocols described in this section have been performed using Candida tropicalis. However it is expected that they will work in the Saccharomycetacaeae Family in general and the Candida genus in particular without undue experimentation since the methods rely upon homologous recombination which is found throughout this Family.

7.1.1. Preparation of DNA Targeting Constructs Prior to Integration into Candida tropicalis

A linear segment of DNA of the form shown schematically in either FIG. 4 or FIG. 7 was prepared by digesting between 2.5 and 5 μg of the plasmid containing the targeting construct with flanking restriction enzymes, in the examples below the restriction enzyme BsmBI from New England Biolabs was used according to the manufacturer's instructions. The digest was purified using Qiagen's PCR purification kit, eluted in 75 μl of Qiagen's EB buffer (elution buffer) and transformed into C. tropicalis by electroporation.

7.1.2. Preparation of Electrocompetent Candida tropicalis

The desired C. tropicalis strain was densely streaked from a culture stored at −80° C. in growth media (YPD) containing 10% glycerol, onto 2-3 100 mm YPD Agar plates and incubated overnight at 30° C. The next morning 10 ml YPD broth was spread onto the surface of the YPD agar plates and the yeast cells were scraped from the plates with the aid of a sterile glass spreader. Cells (of the same strain) from the 2-3 plates were combined in a 50 ml conical tube, and the A₆₀₀ of a 1:20 dilution determined. Sufficient cells to prepare 50 ml of YPD containing yeast cells at an A₆₀₀ of 0.2 were placed in each of two 50 ml conical tubes and pelleted in a centrifuge for 5 min at 400×g. The cells in each tube were suspended in 10 ml of TE/Li mix (100 mM LiCl, 10 mM Tris-Cl, 1 mM EDTA, pH 7.4). Both tubes were incubated in a shaking incubator for 1 hour at 30° C. and 125 rpm, then 250 μl of 1M DTT was added to each 10 ml cell suspension and incubation continued for a further 30 min at 30° C. and 125 rpm.

The cells were then washed twice in water and once in sorbitol. Sterile, ice-cold purified water (40 ml) was added to each of the cell suspensions which were then centrifuged for 5 min at 400×g at 4° C. and the supernatant decanted off. The cells in each tube were resuspended in 50 ml of sterile, ice-cold purified water, centrifuged for 5 min at 400×g at 4° C., the supernatant decanted off supernatant. The cells in each tube were then resuspended in 25 ml of ice cold 1 M Sorbitol (prepared with purified water) and centrifuged for 5 min at 400×g. The supernatant was decanted from each tube and cells resuspended in the small residual volume of Sorbitol solution (the volume of each suspension was approximately 200 μl). The cell suspensions from both tubes were then pooled, this provided enough cells for 4-8 electroporations. In a 1.5 ml eppendorf tube on ice, 60 μl of cells were mixed with 60 μl (˜2.5 μg) of BsmBI digested vector DNA containing the genomic targeting construct. A No DNA Control was prepared for every transformation by mixing cells with Qiagen EB (elution buffer) instead of DNA. The cell-DNA mixtures were mixed with a vortexer and transferred to an ice-cold Bio-Rad 0.2 cm electrode gap Gene Pulser cuvette. The cells were then electroporated at 1.8 kV using a Bio-Rad E. coli Pulser, 1 ml of 1M D-Sorbitol was added and the electroporated cells were transferred to a 14 ml culture tube and 1 ml of 2×YPD broth was added. Cells were then rolled on a Rollerdrum for 1 hour at 37° C. before spreading 100 ul on 100 mm diameter plates containing YPD Agar+200 μg/ml nourseothricin. Plates were incubated for 2-4 days at 30° C. Large colonies (8-16) were individually streaked onto a YPD Agar plate to purify. A single colony from each streak was patched to a YPD agar stock plate and incubated overnight at 30° C.

7.1.3. Genomic DNA Preparation and PCR Test for Integration of Genomic Targeting Constructs at the Desired Location in Candida tropicalis

Between 5 and 30 nourseothricin-resistant isolates were each inoculated into 2 ml of YP Broth and rolled overnight at 30° C. on a Rollerdrum. Genomic DNA from a 0.5 ml sample of each culture was isolated using Zymo Research's YeaStar genomic DNA isolation kit according to the manufacturer's instructions, eluting the DNA in 120 μl of TE, pH 8.0.

For PCR tests, 2.5 μl of the resulting gDNA was used in a 50 ul PCR amplification reaction. As a control for each analysis, genomic DNA was prepared from the parental strain that was transformed with the targeting construct. Oligonucleotide primers for PCR analysis were chosen to lie within the targeting construct and/or in the genomic sequence surrounding the desired integration location, as shown for example in FIG. 10. The size of amplicons was used to determine which strain(s) possessed the desired genomic structure. PCR primer sequences and diagnostic amplicon sizes are described for many of the targeting constructs in Section 7. PCR reaction mixes were prepared containing 5 μl of 10×NEB Standard Taq Buffer, 2.5 μl of dNTP mix (6 mM of each of dATP, dCTP, dGTP, dTTP), 2.5 μl of oligonucleotide primer 1 (10 mM), 2.5 μl of oligonucleotide primer 2 (10 mM), 1 μl of NEB Taq DNA polymerase (5U of enzyme), 2.5 μl of Candida gDNA and water to 50 PCR reactions were subjected to the following temperatures for the times indicated to amplify the target DNA:

Step 1: 1.5 min @ 95° C.

Step 2: 30 sec @ 95° C.

Step 3: 30 sec @ 48° C. (or ˜5° C. lower than the calculated Tm for the primers as appropriate)

Step 4: 1 min @ 72° C. (or 1 minute per 1 kb for predicted amplicon size)

Step 5: Go to step 2 a further 29 times

Step 6: 2 min @ 72° C.

Step 7: Hold @ 4° C.

Step 8: End

The amplicon sizes were determined by running 5-10 μl of the completed PCR reaction on a 1% Agarose-TBE gel.

7.1.4. Selection and Screen for Isolates Having Excised Targeting Constructs from the Genome of Candida tropicalis

Strains carrying a genomic targeting construct to be excised were inoculated from a YPD agar stock plate into 2 ml YP (YPD without dextrose) broth+2% maltose in a 14 ml culture tube. The culture tubes were rolled for ˜48 hours at 30° C. on a rollerdrum. Growth with maltose induced production of Flp recombinase in the host strain from the integrated targeting construct. The Flp recombinase then acted at Frt sites located near the ends of the targeting construct (between the targeting sequences) to excise the sequences between the Frt sites, including the genes encoding Flp recombinase and conferring nourseothricin resistance. The culture was then diluted in serial 10-fold dilutions from 10-fold to 10,000-fold. Aliquots (100 μl) of 100, 1,000 and 10,000-fold dilutions were spread onto YPD agar plates.

Putative excisants were identified by replica-plating colonies on the YPD agar plates from the dilution series (the most useful plates for this purpose were those with 50-500 colonies) to a YPD agar+200 ug/ml nourseothricin plates and then to a YPD agar plate. Putative excisants were identified as colonies that grow on YPD agar, but not YPD agar+200 ug/ml nourseothricin following overnight incubation at room temperature. Putative excisants were streaked for single colonies to a YPD agar plate and incubated overnight at 30 C. A single isolate of each of the putative excisants is patched to a YPD agar stock plate and incubated overnight at 30° C.

Putative excisants were inoculated from the stock plate to 2 ml of YPD broth in a 14 ml culture tube and rolled overnight at 30° C. on a Rollerdrum. Genomic DNA was prepared from 0.5 ml of the overnight culture using the YeaStar Genomic DNA Isolation Kit from Zymo Research and eluted in 120 ul of TE, pH 8.0. Excision of the targeting construct was tested by PCR as described in 7.1.3.

7.2. Deletion of Cytochrome P450 Genes from Candida

The CYP52A type P450s are responsible for oxidation of a variety of compounds in several Candida species, including ω-hydroxylation of fatty acids (Craft et al., 2003, Appl Environ Microbiol: 69, 5983-91; Eschenfeldt et al., 2003, Appl Environ Microbiol: 69, 5992-9; Ohkuma et al., 1991, DNA Cell Biol: 10, 271-82; Zimmer et al., 1995, DNA Cell Biol: 14, 619-28; and Zimmer et al., 1996, Biochem Biophys Res Commun: 224, 784-9.) They have also been implicated in the further oxidation of these compounds. See Eschenfeldt et al., 2003, “Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis.” Appl. Environ. Microbiol. 69: 5992-5999, which is hereby incorporated by reference herein. In some embodiments it is desirable to engineer one or more CYP52A type P450s in a strain of Candida in order to modify the activity or specificity of the P450 enzyme. In some such embodiments it is advantageous to eliminate the activities of one or more CYP52A type P450 enzymes endogenous to the strain. Reasons to delete endogenous P450 enzymes include more accurate determination of the activity and specificity of a P450 enzyme that is being engineered and elimination of P450 enzymes whose activities may interfere with synthesis of the desired product. Strains lacking one or more of their natural CYP52A P450s are within the scope of the disclosed technology. For example in order to obtain a strain of Candida species of yeast including Candida tropicalis for the production of oxidized compounds including ω-hydroxy fatty acids, one method is to reduce or eliminate CYP52A type P450s and other enzyme activities within the cell-that oxidise ω-hydroxy fatty acids to α,ω-diacids. It is then possible to re-introduce one CYP52A type P450 or other enzyme that performs the desired reaction, and to engineer it so that its activity is increased towards desired substrates and reduced towards undesired substrates. In one embodiment its activity for ω-hydroxylation of fatty acids is increased relative to its oxidation of ω-hydroxy fatty acids to α,ω-diacids, thereby favoring the production of ω-hydroxy fatty acids over α,ω-diacids.

7.2.1. Deletion of CYP52A17

The sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A17 is given as SEQ ID NO: 2. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A17 pre-targeting construct is given as SEQ ID NO: 3. Not shown in SEQ ID NO: 3 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of CYP52A17 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the CYP52A17 pre-targeting construct (SEQ ID NO: 3) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of CYP52A17 is given as SEQ ID NO: 4. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of CYP52A17 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 4 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 4 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP65 was prepared by integration of the construct shown as SEQ ID NO: 4 into the genome of strain DP1 (Table 3) at the site of the genomic sequence of the gene for CYP52A17. Candida tropicalis strain DP78 was prepared by excision of the targeting construct from the genome of strain DP65, thereby deleting the gene encoding CYP52A17. Integration and deletion of targeting sequence SEQ ID NO: 4, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were:

17-IN-L3: (SEQ ID NO: 77) TGGCGGAAGTGCATGTGACACAACG 17-IN-R2: (SEQ ID NO: 78) GTGGTTGGTTTGTCTGAGTGGAGAG SAT1-R: (SEQ ID NO: 79) TGGTACTGGTTCTCGGGAGCACAGG SAT1-F: (SEQ ID NO: 80) CGCTAGACAAATTCTTCCAAAAATTTTAGA

For strain DP65 (integration of SEQ ID NO: 4), PCR with primers 17-IN-L3 and SAT1-R produces a 959 base pair amplicon; PCR with primers SAT1-F and 17-IN-R2 produces a 922 base pair amplicon. PCR with primers 17-IN-L3 and 17-IN-R2 from a strain carrying a wild type copy of CYP52A17 produces a 2,372 bae pair amplicon. For strain DP78, with a deleted copy of CYP52A17, PCR with primers 17-IN-L3 and 17-IN-R2 produces a 1,478 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for CYP52A17 will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.2.2. Deletion of CYP52A13

The sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A13 is given as SEQ ID NO: 5. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A13 pre-targeting construct is given as SEQ ID NO: 6. Not shown in SEQ ID NO: 6 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of CYP52A13 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the CYP52A13 pre-targeting construct (SEQ ID NO: 6) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of CYP52A13 is given as SEQ ID NO: 7. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pair of the genomic sequence of CYP52A13 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 7 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 7 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP107 was prepared by integration of the construct shown as SEQ ID NO: 7 into the genome of strain DP65 (Table 3) at the site of the genomic sequence of the gene for CYP52A13. Candida tropicalis strain DP113 was prepared by excision of the targeting construct from the genome of strain DP107, thereby deleting the gene encoding CYP52A13. Integration and deletion of targeting sequence SEQ ID NO: 7, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

13-IN-L2: (SEQ ID NO: 81) CATGTGGCCGCTGAATGTGGGGGCA 13-IN-R2: (SEQ ID NO: 82) GCCATTTTGTTTTTTTTTACCCCTCTAACA SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP107 (integration of SEQ ID NO: 7), PCR with primers 13-IN-L2 and SAT1-R produces an 874 base pair amplicon; PCR with primers SAT1-F and 13-IN-R2 produces an 879 base pair amplicon. PCR with primers 13-IN-L2 and 13-IN-R2 from a strain with wild type CYP52A13 produces a 2,259 base pair amplicon. For strain DP113 with a deleted version of CYP52A13 PCR with primers 13-IN-L2 and 13-IN-R2 produces a 1,350 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for CYP52A13 will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.2.3. Deletion of CYP52A18

The sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A18 is given as SEQ ID NO: 8. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A18 pre-targeting construct is given as SEQ ID NO: 9. The CYP52A18 pre-targeting construct also contains a polylinker sequence (SEQ ID NO: 10) between the 5′ targeting sequence and the NotI site. This polylinker sequence was placed to allow the insertion of sequences into the targeting construct to allow it to function as an insertion targeting construct of the form shown schematically in FIG. 7. Not shown in SEQ ID NO: 9 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art. A targeting construct for deletion of CYP52A18 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the CYP52A18 pre-targeting construct (SEQ ID NO: 9) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of CYP52A18 is given as SEQ ID NO: 11. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of CYP52A18 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 11 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 11 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP140 was prepared by integration of the construct shown as SEQ ID NO: 11 into the genome of strain DP113 (Table 3) at the site of the genomic sequence of the gene for CYP52A18. Candida tropicalis strain DP142 was prepared by excision of the targeting construct from the genome of strain DP140, thereby deleting the gene encoding CYP52A18. Integration and deletion of targeting sequence SEQ ID NO: 11, and analysis of integrants and excisants were performed as described in Section 7.1.

Oligonucleotide primers for analysis of strains were:

18-IN-L2: (SEQ ID NO: 83) GGAAGTGCATGTGACACAATACCCT 18-IN-R2: (SEQ ID NO: 84) GGTGGTTTGTCTGAGTGAGAACGTTTAATT SAT1-R: (SEQ ID NO: 79) TGGTACTGGTTCTCGGGAGCACAGG SAT1-F: (SEQ ID NO: 80) CGCTAGACAAATTCTTCCAAAAATTTTAGA

For strain DP140 (integration of SEQ ID NO: 11), PCR with primers 18-IN-L2 and SAT1-R produces a 676 base pair amplicon; PCR with primers SAT1-F and 18-IN-R2 produces a 605 base pair amplicon. PCR from a strain with a wild type version of CYP52A18 with primers 18-IN-L2 and 18-IN-R2 produces a 2,328 base pair amplicon. For strain DP142 with a deleted version of CYP52A18, PCR with primers 18-IN-L2 and 18-IN-R2 produces an 878 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for CYP52A18 will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.2.4. Deletion of CYP52A14

The sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A14 is given as SEQ ID NO: 13. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A14 pre-targeting construct is given as SEQ ID NO: 14. The CYP52A14 pre-targeting construct also contains a polylinker sequence (SEQ ID NO: 10) between the 5′ targeting sequence and the NotI site. This polylinker sequence was placed to allow the insertion of sequences into the targeting construct to allow it to function as an insertion targeting construct of the form shown schematically in FIG. 7. Not shown in SEQ ID NO: 14 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of CYP52A14 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the CYP52A14 pre-targeting construct (SEQ ID NO: 14) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of CYP52A14 is given as SEQ ID NO: 15. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of CYP52A14 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 15 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 15 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP170 was prepared by integration of the construct shown as SEQ ID NO: 15 into the genome of strain DP142 (Table 3) at the site of the genomic sequence of the gene for CYP52A14. Candida tropicalis strain DP174 was prepared by excision of the targeting construct from the genome of strain DP170, thereby deleting the gene encoding CYP52A14. Integration and deletion of targeting sequence SEQ ID NO: 15, and analysis of integrants and excisants were performed as described in Section 7.1.

Oligonucleotide primers for analysis of strains were:

14-IN-L2: (SEQ ID NO: 85) GACGTAGCCGATGAATGTGGGGTGC 14-IN-R2: (SEQ ID NO: 86) TGCCATTTATTTTTTATTACCCCTCTAAAT SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP170 (integration of SEQ ID NO: 15), PCR with primers 14-IN-L2 and SAT1-R produces a 664 base pair amplicon; PCR with primers SAT1-F and 14-IN-R2 produces a 609 base pair amplicon. For a strain with a wild type version of CYP52A14, PCR with primers 14-IN-L2 and 14-IN-R2 produces a 2,234 base pair amplicon. For strain DP174 with a deleted version of CYP52A14, PCR with primers 14-IN-L2 and 14-IN-R2 produces an 870 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for CYP52A14 will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.3. Deletion of Fatty Alcohol Oxidase Genes from Candida

At least one enzyme capable of oxidizing ω-hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18. Oxidation of energy rich molecules reduces their energy content. For the production of incompletely oxidized compounds-including ω-hydroxy fatty acids, it is advantageous to reduce or eliminate the further oxidation of incompletely oxidized compounds-such as ω-hydroxy fatty acids. Under one aspect, this can be achieved by deleting the genes encoding the oxidizing enzymes from the Candida genome, Candidate genes for this activity include fatty alcohol oxidase and dehydrogenases as shown in FIG. 14. One class of enzymes known to oxidize incompletely oxidised compounds including hydroxy fatty acids are the fatty alcohol oxidases.

7.3.1. Deletion of FAO1

The sequence of a gene encoding a fatty alcohol oxidase in Candida tropicalis, FAO1 is given as SEQ ID NO: 16. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the FAO1 pre-targeting construct is given as SEQ ID NO: 17. The FAO1 pre-targeting construct also contains a polylinker sequence (SEQ ID NO: 10) between the 5′ targeting sequence and the NotI site. This polylinker sequence was placed to allow the insertion of sequences into the targeting construct to allow it to function as an insertion targeting construct of the form shown schematically in FIG. 7. Not shown in SEQ ID NO: 17 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of FAO1 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the FAO1 pre-targeting construct (SEQ ID NO: 17) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of FAO1 is given as SEQ ID NO: 18. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of FAO1 at the 5′ end and 220 base pairs of the genomic sequence of FAO1 at the 3′ end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 18 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 18 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP182 was prepared by integration of the construct shown as SEQ ID NO: 18 into the genome of strain DP174 (Table 3) at the site of the genomic sequence of the gene for FAO1. Candida tropicalis strain DP186 was prepared by excision of the targeting construct from the genome of strain DP182, thereby deleting the gene encoding FAO1. Integration and deletion of targeting sequence SEQ ID NO: 18, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

FAO1-IN-L: (SEQ ID NO: 87) ATTGGCGTCGTGGCATTGGCGGCTC FAO1-IN-R: (SEQ ID NO: 88) TGGGCGGAATCAAGTGGCTT SAT1-R: (SEQ ID NO: 79) TGGTACTGGTTCTCGGGAGCACAGG SAT1-F: (SEQ ID NO: 80) CGCTAGACAAATTCTTCCAAAAATTTTAGA

For strain DP182 (integration of SEQ ID NO: 18), PCR with primers FAO1-IN-L and SAT1-R produces a 624 base pair amplicon; PCR with primers SAT1-F and FAO1-IN-R produces a 478 base pair amplicon. For a strain with a wild type copy of FAO1, PCR with primers FAO1-IN-L and FAO1-IN-R produces a 2,709 base pair amplicon. For strain DP186 with a deleted copy of FAO1, PCR with primers FAO1-IN-L and FAO1-IN-R produces a 699 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for FAO1A will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.3.2. Deletion of FAO1B

No sequence had been reported for a second allele for FAO1 (FAO1B) at the time of this work. To identify the allele (BAO1B) we used PCR amplification primers and sequencing primers designed to anneal to the known sequenced allele of FAO1. The primers used were:

FAO1_F1; CGTCGACACCCTTATGTTAT (SEQ ID NO: 89) FAO1_F2; CGTTGACTCCTATCAAGGACA (SEQ ID NO: 90) FAO1_R1; GGTCTTCTCTTCCTGGATAATG (SEQ ID NO: 91) FAO1_F3; CCAGCAGTTGTTTGTTCTTG (SEQ ID NO: 92) FAO1_R2; AATCCTGTGCTTTGTCGTAGGC (SEQ ID NO: 93) FAO1_F4; TCCTTAACAAGAAGGGCATCG (SEQ ID NO: 94) FAO1_R3; TTCTTGAATCCGGAGTTGAC (SEQ ID NO: 95) FAO1_F5; TCTTAGTCGTGATACCACCA (SEQ ID NO: 96) FAO1_R4; CTAAGGATTCTCTTGGCACC (SEQ ID NO: 97) FAO1_R5; GTGACCATAGGATTAGCACC (SEQ ID NO: 98)

Genomic DNA was prepared from strains DP1 (which has FAO1) and DP186 (which is deleted for FAO1) as described in section 7.1.3. The FAO genes were amplified from genomic DNA by PCR using oligonucleotide primers FAO1_F1 and FAO1_R5. Genomic DNA from both strains yielded an amplicon of approximately 2 kilobases. Both amplicons were directly sequenced using the ten oligonucleotide primers listed above. The amplicon from DP1 gave sequence where there were occasionally two bases that appeared to be equally represented. The amplicon from DP186 had no such ambiguous bases but its sequence was slightly different (˜95% identical) from the reported sequence of FAO1. We concluded that the sequence corresponded to a second allele of FAO1, which we refer to as FAO1B. The sequence of FAO1B is given as SEQ ID NO: 19.

This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 bp stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the FAO1B pre-targeting construct is given as SEQ ID NO: 20.

A targeting construct for deletion of FAO1 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the FAO1B pre-targeting construct (SEQ ID NO: 20) that had also been digested with restriction enzymes NotI and XhoI. the FAO1B pre-targeting construct (SEQ ID NO: 20) was not cloned or propagated in a bacterial host, so digestion with restriction enzymes NotI and XhoI produced two fragments which were then ligated with the digested SAT-1 flipper to produce a targeting construct for deletion of FAO1B, given as SEQ ID NO: 21. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of FAO1B at the 5′ end and 220 base pairs of the genomic sequence of FAO1B at the 3′ end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.

Candida tropicalis strain DP238 was prepared by integration of the construct shown as SEQ ID NO: 21 into the genome of strain DP186 (Table 3) at the site of the genomic sequence of the gene for FAO1B. Candida tropicalis strain DP240 was prepared by excision of the targeting construct from the genome of strain DP238, thereby deleting the gene encoding FAO1B. Integration and deletion of targeting sequence SEQ ID NO: 21, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were, FAO1_F1 (SEQ ID NO: 89), FAO1_R5 (SEQ ID NO: 98), SAT1-R (SEQ ID NO: 79), SAT1-F (SEQ ID NO: 80).

For strain DP182 (integration of SEQ ID NO: 18), PCR with primers FAO1_F1 and SAT1-R produces a 558 base pair amplicon; PCR with primers SAT1-F and FAO1_R5 produces a 557 base pair amplicon. For a strain with a wild type copy of FAO1B, PCR with primers FAO1_F1 and FAO1_R5 produces a 2,007 base pair amplicon. For strain DP186, with a deleted copy of FAO1B, PCR with primers FAO1_F1 and FAO1_R5 produces a 711 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for FAO1B will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.3.3. Deletion of FAO2A

The sequence of a gene encoding a fatty alcohol oxidase in Candida tropicalis, FAO2A is given as SEQ ID NO: 22. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 by stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the FAO2A pre-targeting construct is given as SEQ ID NO: 23. Not shown in SEQ ID NO: 23 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of FAO2A from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the FAO2A pre-targeting construct (SEQ ID NO: 23) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of FAO2A is given as SEQ ID NO: 24. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pair of the genomic sequence of FAO2A at the 5′ and 3′ ends of the structural gene to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 24 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 24 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP255 was prepared by integration of the construct shown as SEQ ID NO: 24 into the genome of strain DP240 (Table 3) at the site of the genomic sequence of the gene for FAO2A. Candida tropicalis strain DP256 was prepared by excision of the targeting construct from the genome of strain DP255, thereby deleting most of the coding portion of the gene encoding FAO2A. Integration and deletion of targeting sequence SEQ ID NO: 24, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were:

FAO2A-IN-L: (SEQ ID NO: 99) CTTTTCTGATTCTTGATTTTCCCTTTTCAT FAO2A-IN-R: (SEQ ID NO: 100) ATACATCTAGTATATAAGTGTCGTATTTCC SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP255 (integration of SEQ ID NO: 24), PCR with primers FAO2A-IN-L and SAT1-R produces a 581 base pair amplicon; PCR with primers SAT1-F and FAO2A-IN-R produces a 569 base pair amplicon. For a strain with a wild type copy of FAO2A, PCR with primers FAO2A-IN-L and FAO2A-IN-R produces a 2,199 base pair amplicon. For strain DP186 with a deleted copy of FAO2A, PCR with primers FAO2A-IN-L and FAO2A-IN-R produces a 747 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for FAO2A will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.3.4. Deletion of FAO2B

The sequence of a gene encoding a fatty alcohol oxidase in Candida tropicalis, FAO2B is given as SEQ ID NO: 25. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the FAO2B pre-targeting construct is given as SEQ ID NO: 26. Not shown in SEQ ID NO: 26 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of FAO2B from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the FAO2B pre-targeting construct (SEQ ID NO: 26) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of FAO2B is given as SEQ ID NO: 27. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of FAO2B at the 5′ and 3′ ends of the structural gene to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 27 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 27 also includes a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP259 was prepared by integration of the construct shown as SEQ ID NO: 27 into the genome of strain DP256 (Table 3) at the site of the genomic sequence of the gene for FAO2BA. Candida tropicalis strain DP261 was prepared by excision of the targeting construct from the genome of strain DP259, thereby deleting most of the coding region of the gene encoding FAO2B. Integration and deletion of targeting sequence SEQ ID NO: 27, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

FAO2B-IN-L: (SEQ ID NO: 101) TGCTTTTCTGATTCTTGATCATCCCCTTAG FAO2B-IN-R: (SEQ ID NO: 102) ATACATCTAGTATATAAGTGTCGTATTTCT SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP259 (integration of SEQ ID NO: 27), PCR with primers FAO2B-IN-L and SAT1-R produces a 551 base pair amplicon; PCR with primers SAT1-F and FAO2B-IN-R produces a 571 base pair amplicon. For a strain with a wild type copy of FAO2B, PCR with primers FAO2B-IN-L and FAO2B-IN-R produces a 2,198 base pair amplicon. For strain DP186 with a deleted copy of FAO2B, PCR with primers FAO2B-IN-L and FAO2B-IN-R produces a 719 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for FAO2B will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.4. Deletion of More Cytochrome P450 Genes from Candida

At least one enzyme capable of oxidizing ω-hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18 and fatty alcohol oxidase genes FAO1, FAO1B, FAO2A and FAO2B. Oxidation of energy rich molecules reduces their energy content. For the production of incompletely oxidized compounds-including ω-hydroxy fatty acids, it is advantageous to reduce or eliminate the further oxidation of incompletely oxidized compounds ω-hydroxy fatty acids. Under one aspect, this can be achieved by deleting the genes encoding the oxidizing enzymes from the Candida genome. One class of enzymes known to oxidize incompletely oxidised compounds are the cytochroime P450s.

The CYP52A type P450s are responsible for ω-hydroxylation of fatty acids in several Candida species (Craft et al., 2003, Appl Environ Microbiol: 69, 5983-91; Eschenfeldt et al., 2003, Appl Environ Microbiol: 69, 5992-9; Ohkuma et al., 1991, DNA Cell Biol: 10, 271-82; Zimmer et al., 1995, DNA Cell Biol: 14, 619-28; Zimmer et al., 1996, Biochem Biophys Res Commun: 224, 784-9.) They have also been implicated in the further oxidation of these ω-hydroxy fatty acids to α,ω-diacids. See Eschenfeldt, et al., 2003, Appli. Environ. Microbiol. 69: 5992-5999, which is hereby incorporated by reference herein. Another CYP52A type P450 whose expression is induced by fatty acids is CYP52A12.

7.4.1. Deletion of CYP52A12

The sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A12 is given as SEQ ID NO: 28. This sequence was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and a XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A12 pre-targeting construct is given as SEQ ID NO: 29. Not shown in SEQ ID NO: 29 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of CYP52A12 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the CYP52A12 pre-targeting construct (SEQ ID NO: 29) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of CYP52A12 is given as SEQ ID NO: 30. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of CYP52A12 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 30 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 30 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP268 was prepared by integration of the construct shown as SEQ ID NO: 30 into the genome of strain DP261 (Table 3) at the site of the genomic sequence of the gene for CYP52A12. Candida tropicalis strain DP272 was prepared by excision of the targeting construct from the genome of strain DP268, thereby deleting the gene encoding CYP52A12. Integration and deletion of targeting sequence SEQ ID NO: 30, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

12-IN-L: (SEQ ID NO: 103) CGCCAGTCTTTCCTGATTGGGCAAG 12-IN-R2: (SEQ ID NO: 104) GGACGTTGTCGAGTAGAGGGATGTG SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP268 (integration of SEQ ID NO: 30), PCR with primers 12-IN-L and SAT1-R produces a 596 base pair amplicon; PCR with primers SAT1-F and 12-IN-R2 produces a 650 base pair amplicon. For a strain with a wild type copy of CYP52A12, PCR with primers 12-IN-L and 12-IN-R2 produces a 2,348 base pair amplicon. For strain DP272 with a deleted copy of CYP52A12, PCR with primers 12-IN-L and 12-IN-R2 produces a 843 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for CYP52A12 will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.4.2. Deletion of CYP52A12B

No sequence had been reported for a second allele for CYP52A12 at the time of this work. We reasoned that in a diploid organisms a second allele existed (CYP52A17 and CYP52A18 are an allelic pair and CYP52A13 and CYP52A14 are an allelic pair). To delete the second allele we synthesized a deletion construct based on the CYP52A12 sequence (SEQ ID NO: 28), but designed it so that the targeting sequences were homologous to regions of the CYP52A12 gene that are missing because they have been deleted in strain DP272. First we constructed a “pre-targeting” construct comprising two targeting sequences from near the 5′ and 3′ ends of the structural gene, but internal to the two sequences used in the design of the targeting construct for the deletion of CYP52A12. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and a XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A12B pre-targeting construct is given as SEQ ID NO: 31. Not shown in SEQ ID NO: 31 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of CYP52A12B from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the CYP52A12B pre-targeting construct (SEQ ID NO: 31) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of CYP52A12B is given as SEQ ID NO: 32. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 300 base pairs of the genomic sequence of CYP52A12 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 32 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 32 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP282 was prepared by integration of the construct shown as SEQ ID NO: 32 into the genome of strain DP272 (Table 3) at the site of the genomic sequence of the gene for CYP52A12B. Candida tropicalis strain DP284 was prepared by excision of the targeting construct from the genome of strain DP282, thereby deleting a portion of the coding region of the gene encoding CYP52A12B. Integration and deletion of targeting sequence SEQ ID NO: 32, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

12-F1: CTGTACTTCCGTACTTGACC (SEQ ID NO: 105) 12-R1: GAGACCTGGATCAGATGAG (SEQ ID NO: 106) SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

Oligonucleotides 12-F1 and 12-R1 are designed to anneal to a part of the genome that is missing in strains with deletions in CYP52A12. In such strains they will thus only be able to anneal to and amplify from the second allele CYP52A12B. For strain DP282 (integration of SEQ ID NO: 32), PCR with primers 12-F1 and SAT1-R produces a 978 base pair amplicon; PCR with primers SAT1-F and 12-R1 produces a 947 base pair amplicon. PCR from a strain with a wild type copy of CYP52A12B with primers 12-F1 and 12-R1 produces a 1,478 base pair amplicon. For strain DP272 with a deleted copy of CYP52A12B, PCR with primers 12-F1 and 12-R1 produces a 505 base pair amplicon.

Deletion of a portion of the coding sequence of the gene for CYP52A12B will disrupt the function of the protein encoded by this gene in the Candida host cell.

7.5. Deletion of Alcohol Dehydrogenase Genes from Candida

At least one enzyme capable of oxidizing ω-hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17, CYP52A18, CYP52A12, CYP52A12B and the fatty alcohol oxidase genes FAO1, FAO1B, FAO2A and FAO2B. Oxidation of energy rich molecules reduces their energy content. For the production of incompletely oxidized compounds including ω-hydroxy fatty acids, it is advantageous to reduce or eliminate the further oxidation of incompletely oxidized compounds, including for example ω-hydroxy fatty acids. Under one aspect, this can be achieved by deleting the genes encoding the oxidizing enzymes from the Candida genome. One class of enzymes known to oxidize alcohols is alcohol dehydrogenases.

7.5.1. Identification of Candida tropicalis Alcohol Dehydrogenases

The sequences of four alcohol dehydrogenase genes were obtained from the Candida Geneome Database in the Department of Genetics at the School of Medicine, Stanford University, Palo Alto, Calif. The sequences of these genes are given as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36. These sequences were aligned and two degenerate oligonucleotide primers were designed, whose sequences are given as SEQ ID NO: 37 and SEQ ID NO: 38. These two primers were used to PCR amplify from genomic DNA from Candida tropicalis strain DP1. The resulting amplicon of ˜1,000 base pairs was cloned and 96 independent transformants were picked, plasmid prepared and sequenced using two primers with annealing sites located in the vector reading into the cloning site and two primers designed to anneal to highly conserved sequences within the Candida albicans alcohol dehydrogenase sequences:

ADH-F: GTTTACAAAGCCTTAAAGACT (SEQ ID NO: 107) ADH-R: TTGAACGGCCAAAGAACCTAA. (SEQ ID NO: 108)

Five different sequences were obtained by sequencing the 96 independent clones, called Ct_ADH-A4, Ct_ADH-A10, Ct_ADH-B2, Ct_ADH-B4 and Ct_ADH-B11. These sequences are provided as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively. In silico translation of Ct_ADH-B2 (SEQ ID NO: 41) yielded an amino acid sequence with multiple in-frame stop codons, so it is almost certainly a pseudogene and does not encode a functional protein. The other four sequences all encode protein sequences without stop codons.

Amino acid sequences of the partial genes are predicted and provided: SEQ ID NO:155 (ADH-A4), SEQ ID NO:154 (ADH-B4), SEQ ID NO:152 (ADH-A10), SEQ ID NO:153 (ADH-A10B) and SEQ ID NO:151 (ADH-B11).

In some embodiments an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison with the nucleotide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if (i) it comprises an open reading frame encoding a polypeptide at least 275 amino acids long or at least 300 amino acids long and (ii) the gene is at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical for a stretch of at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 contiguous nucleotides of the coding sequence of a Candida tropicalis gene selected from the group consisting of ADH-A4 (SEQ ID NO: 39), ADH-B4 (SEQ ID NO: 42), ADH-A10 (SEQ ID NO: 40), ADH-A10B (SEQ ID NO: 56), and ADH-B11 (SEQ ID NO: 43).

The sequence relationships of these protein sequences are shown in a phylogenetic tree in FIG. 17. Ct_ADH-A4 (encoded by SEQ ID NO: 39) is most homologous to Candida albicans ADH1A and Ct_ADH-B4 (encoded by SEQ ID NO: 42) is most homologous to Candida albicans ADH2A.

An alignment, using ClustalW, of the amino acid sequences of alcohol dehydrogenase proteins predicted from the sequences of genes from Candida albicans and Candida tropicalis is shown in FIGS. 3A and 3B. The genes from Candida tropicalis were isolated as partial genes by PCR with degenerate primers, so the nucleic acid sequences obtained for the genes represent only a partial sequence of the gene, and the predicted amino acid sequences of the encoded proteins represent only a partial sequence of the protein. A consensus is indicated underneath the aligned amino acid sequences of FIGS. 3A and 3B, with a * indicating that all 4 Candida albicans alcohol dehydrogenase sequences and all 4 Candida tropicalis alcohol dehydrogenase sequences are completely identical at those residues. BLAST searching of protein sequences in Genbank with highly conserved peptide regions within the alcohol dehydrogenases yields results that identify uniquely yeast alcohol dehydrogenases.

In some embodiments an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a first peptide sequence VKYSGVCH (SEQ ID NO: 156) or VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162) or VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163) or VGGHEGAGVVV (SEQ ID NO: 157).

In some embodiments an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a second peptide sequence QYATADAVQAA (SEQ ID NO: 158) or SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164) or GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165). In some embodiments an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a third peptide sequence CAGVTVYKALK (SEQ ID NO: 159) or APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a fourth peptide sequence GQWVAISGA (SEQ ID NO: 160) or GQWVAISGAxGGLGSL (SEQ ID NO: 167) or GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168) or GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169) or GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).

The four coding sequences were sufficiently dissimilar to reach the conclusion that they were not allelic pairs, but rather represented four different genes, each of which probably had its own allelic partner in the genome. Each of the coding sequences was thus used to design two targeting constructs, similarly to the strategy described for CYP52A12B in Section 7.4.2. The construct for the first allele of each ADH gene used ˜200 base pairs at the 5′ end and ˜200 base pairs at the 3′ end as targeting sequences (5′-ADH Out and 3′-ADH Out in FIG. 18). The construct for the second allele used two sections of ˜200 base pairs between the first two targeting sequences (5′-ADH In and 3′-ADH in FIG. 18). These sequences will be eliminated by the first targeting construct from the first allele of the gene and will thus serve as a targeting sequence for the second allele of the gene. As described below, this strategy succeeded with two ADH allelic pairs: those for ADH-A4 and ADH-B4. However at the first attempt it was not successful for deletion of the second allele of ADH-A10 or ADH-B11, so the second allele of these genes were isolated, sequenced and those sequences were used to delete the second alleles of ADH-A10 or ADH-B11.

Deletion of a portion of the sequence of an alcohol dehydrogenase gene will disrupt the function of that alcohol dehydrogenase enzyme in the Candida host cell.

In some embodiments, disruption of an alcohol dehydrogenase in a first host cell organism is measured by incubating the first host cell organism in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The rate of conversion of the substrate by the first host cell organism is compared with the rate of conversion produced by a second host cell organism that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.). The rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is disrupted if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower in the first host cell organism than the second host cell organism.

In some embodiments, disruption of an alcohol dehydrogenase in a first host cell organism is measured by incubating said first host cell organism in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group. The amount of the substrate converted to product by the first host cell organism in a specified time is compared with the amount of substrate converted to product by a second host cell organism that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.). The amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays. In some embodiments the alcohol dehydrogenase is disrupted if the amount of product is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the first host cell organism than the second host cell organism.

7.5.2. Deletion of ADH-A4

Sequence SEQ ID NO: 39 was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the ADH-A4 structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-A4 pre-targeting construct is given as SEQ ID NO: 44. Not shown in SEQ ID NO: 44 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-A4 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-A4 pre-targeting construct (SEQ ID NO: 44) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-A4 is given as SEQ ID NO: 45. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 base pairs of the genomic sequence of ADH-A4 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 44 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 44 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP387 was prepared by integration of the construct shown as SEQ ID NO: 45 into the genome of strain DP283 (Table 3) at the site of the genomic sequence of the gene for ADH-A4. Candida tropicalis strain DP388 was prepared by excision of the targeting construct from the genome of strain DP387, thereby deleting the gene encoding ADH-A4. Integration and deletion of targeting sequence SEQ ID NO: 45, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

A4-OUT-F: (SEQ ID NO: 109) GAATTAGAATACAAAGATATCCCAGTG A4-OUT-R: (SEQ ID NO: 110) CATCAACTTGAAGACCTGTGGCAAT SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP387 (integration of SEQ ID NO: 45), PCR with primers A4-OUT-F and SAT1-R produces a 464 base pair amplicon; PCR with primers SAT1-F and A4-OUT-R produces a 464 base pair amplicon. PCR from a strain with a wild type copy of ADH-A4 with primers A4-OUT-F and A4-OUT-R produces a 948 base pair amplicon. For strain DP388 with a deleted copy of ADH-A4, PCR with primers A4-OUT-F and A4-OUT-R produces a 525 base pair amplicon.

7.5.3. Deletion of ADH-A4B

No sequence was identified for a second allele for ADH-A4 in the initial set of 96 sequences but we reasoned that in a diploid organism, a second allele existed. To delete the second allele (ADH-A4B) we synthesized a deletion construct based on the ADH-A4 sequence (SEQ ID NO: 39), but designed it so that the targeting sequences were homologous to regions of the ADH-A4 gene that are missing because they have been deleted in strain DP388. First we constructed a “pre-targeting” construct comprising two targeting sequences internal to the two sequences used in the design of the targeting construct for the deletion of ADH-A4. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-A4B pre-targeting construct is given as SEQ ID NO: 46. Not shown in SEQ ID NO: 46 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-A4B from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-A4B pre-targeting construct (SEQ ID NO: 46) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-A4B is given as SEQ ID NO: 47. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 base pairs of the genomic sequence of ADH-A4B at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 47 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 47 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP389 was prepared by integration of the construct shown as SEQ ID NO: 47 into the genome of strain DP388 (Table 3) at the site of the genomic sequence of the gene for ADH-A4B. Candida tropicalis strain DP390 was prepared by excision of the targeting construct from the genome of strain DP389, thereby deleting a portion of the coding region of the gene encoding ADH-A4B. Integration and deletion of targeting sequence SEQ ID NO: 47, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

A4-IN-F: (SEQ ID NO: 111) GAACGGTTCCTGTATGTCCTGTGAGTT A4-IN-R: (SEQ ID NO: 112) CGGATTGGTCAATGGCTTTTTCGGAA SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

Oligonucleotides A4-IN-F and A4-IN-R are designed to anneal to a part of the genome that is missing in strains with deletions in ADH-A4. In such strains they will thus only be able to anneal to and amplify from the second allele ADH-A4B. For strain DP389 (integration of SEQ ID NO: 47), PCR with primers A4-IN-F and SAT1-R produces a 462 base pair amplicon; PCR with primers SAT1-F and A4-IN-R produces a 462 base pair amplicon. PCR from a strain with a wild-type copy of ADH-A4B with primers A4-IN-F and A4-IN-R produces a 488 base pair amplicon. For strain DP390 with a deleted copy of ADH-A4B, PCR with primers A4-IN-F and A4-IN-R produces a 521 base pair amplicon. The amplicons with primers A4-IN-F and A4-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH-A4B, but digestion of the amplicon with either NotI or XhoI will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.

7.5.4. Deletion of ADH-B4

Sequence SEQ ID NO: 42 was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the ADH-B4 structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 by stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-B4 pre-targeting construct is given as SEQ ID NO: 48. Not shown in SEQ ID NO: 48 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-B4 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-B4 pre-targeting construct (SEQ ID NO: 48) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-B4 is given as SEQ ID NO: 49. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 by of the genomic sequence of ADH-B4 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 49 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 49 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP397 was prepared by integration of the construct shown as SEQ ID NO: 49 into the genome of strain DP390 (Table 3) at the site of the genomic sequence of the gene for ADH-B4. Candida tropicalis strain DP398 was prepared by excision of the targeting construct from the genome of strain DP397, thereby deleting the gene encoding ADH-B4. Integration and deletion of targeting sequence SEQ ID NO: 49, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

B4-OUT-F: (SEQ ID NO: 113) AAATTAGAATACAAGGACATCCCAGTT B4-OUT-R: (SEQ ID NO: 114) CATCAACTTGTAGACTTCTGGCAAT SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP397 (integration of SEQ ID NO: 49), PCR with primers B4-OUT-F and SAT1-R produces a 464 by amplicon; PCR with primers SAT1-F and B4-OUT-R produces a 464 base pair amplicon. PCR from a strain with a wild type copy of ADH-B4 with primers B4-OUT-F and B4-OUT-R produces a 948 base pair amplicon. For strain DP398 with a deleted copy of ADH-B4, PCR with primers B4-OUT-F and B4-OUT-R produces a 525 base pair amplicon.

7.5.5. Deletion of ADH-B4B

No sequence was identified for a second allele for ADH-B4 in the initial set of 96 sequences but we reasoned that in a diploid organism a second allele existed. To delete the second allele (ADH-B4B) we synthesized a deletion construct based on the ADH-B4 sequence (SEQ ID NO: 42), but designed it so that the targeting sequences were homologous to regions of the ADH-B4 gene that are missing because they have been deleted in strain DP398. First we constructed a “pre-targeting” construct comprising two targeting sequences internal to the two sequences used in the design of the targeting construct for the deletion of ADH-B4. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-B4B pre-targeting construct is given as SEQ ID NO: 50. Not shown in SEQ ID NO: 50 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-B4B from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-B4B pre-targeting construct (SEQ ID NO: 50) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-B4B is given as SEQ ID NO: 51. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 by of the genomic sequence of ADH-B4B at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 51 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 51 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP409 was prepared by integration of the construct shown as SEQ ID NO: 51 into the genome of strain DP398 (Table 3) at the site of the genomic sequence of the gene for ADH-B4B. Candida tropicalis strain DP411 was prepared by excision of the targeting construct from the genome of strain DP409, thereby deleting a portion of the coding region of the gene encoding ADH-B4B. Integration and deletion of targeting sequence SEQ ID NO: 51, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

B4-IN-F: (SEQ ID NO: 115) GAACGGTTCCTGTATGAACTGTGAGTA B4-IN-R: (SEQ ID NO: 116) CAGATTGGTTGATGGCCTTTTCGGAG SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

Oligonucleotides B4-IN-F and B4-IN-R are designed to anneal to a part of the genome that is missing in strains with deletions in ADH-B4. In such strains they will thus only be able to anneal to and amplify from the second allele ADH-B4B. For strain DP409 (integration of SEQ ID NO: 51), PCR with primers B4-IN-F and SAT1-R produces a 462 base pair amplicon; PCR with primers SAT1-F and B4-IN-R produces a 462 base pair amplicon. PCR from a strain with a wild-type copy of ADH-B4B with primers B4-IN-F and B4-IN-R produces a 488 base pair amplicon. For strain DP411 with a deleted copy of ADH-B4B, PCR with primers B4-IN-F and B4-IN-R produces a 521 base pair amplicon. The amplicons with primers B4-IN-F and B4-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH-B4B, but digestion of the amplicon with either NotI or XhoI will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.

7.5.6. Deletion of ADH-A10

Sequence SEQ ID NO: 40 was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the ADH-A10 structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 by stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-A10 pre-targeting construct is given as SEQ ID NO: 52. Not shown in SEQ ID NO: 52 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-A10 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-A10 pre-targeting construct (SEQ ID NO: 52) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-A10 is given as SEQ ID NO: 53. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 by of the genomic sequence of ADH-A10 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 53 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 53 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP415 was prepared by integration of the construct shown as SEQ ID NO: 53 into the genome of strain DP411 (Table 3) at the site of the genomic sequence of the gene for ADH-A10. Candida tropicalis strain DP416 was prepared by excision of the targeting construct from the genome of strain DP415, thereby deleting the gene encoding ADH-A10. Integration and deletion of targeting sequence SEQ ID NO: 53, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

A10-OUT-F: (SEQ ID NO: 117) AAGTTAGAATACAAAGACGTGCCGGTC A10-OUT-R: (SEQ ID NO: 118) CATCAAGTCAAAAATCTCTGGCACT SAT1-R: (SEQ ID NO: 147) SAT1-F: (SEQ ID NO: 80)

For strain DP415 (integration of SEQ ID NO: 49), PCR with primers A10-OUT-F and SAT1-R produces a 464 base pair amplicon; PCR with primers SAT1-F and A10-OUT-R produces a 464 base pair amplicon. PCR from a strain with a wild type copy of ADH-A10 with primers A10-OUT-F and A10-OUT-R produces a 948 base pair amplicon. For strain DP416 with a deleted copy of ADH-A10, PCR with primers A10-OUT-F and A10-OUT-R produces a 525 base pair amplicon.

7.5.7. Deletion of ADH-B11

Sequence SEQ ID NO: 43 was used to design a “pre-targeting” construct comprising two targeting sequences from the 5′ and 3′ end of the ADH-B11 structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-B11 pre-targeting construct is given as SEQ ID NO: 54. Not shown in SEQ ID NO: 54 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-B11 from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-B11 pre-targeting construct (SEQ ID NO: 54) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-B11 is given as SEQ ID NO: 55. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 base pair of the genomic sequence of ADH-B11 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 55 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 53 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP417 was prepared by integration of the construct shown as SEQ ID NO: 55 into the genome of strain DP416 (Table 3) at the site of the genomic sequence of the gene for ADH-B11. Candida tropicalis strain DP421 was prepared by excision of the targeting construct from the genome of strain DP417, thereby deleting the gene encoding ADH-B11. Integration and deletion of targeting sequence SEQ ID NO: 55, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

B11-OUT-F: (SEQ ID NO: 119) CCATTGCAATACACCGATATCCCAGTT B11-OUT-R: (SEQ ID NO: 120) CAACAATTTGAAAATCTCTGGCAAT SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP417 (integration of SEQ ID NO: 49), PCR with primers B11-OUT-F and SAT1-R produces a 464 base pair amplicon; PCR with primers SAT1-F and B11-OUT-R produces a 464 base pair amplicon. PCR from a strain with a wild type copy of ADH-B11 with primers B11-OUT-F and B11-OUT-R produces a 948 base pair amplicon. For strain DP421 with a deleted copy of ADH-B11, PCR with primers B11-OUT-F and B11-OUT-R produces a 525 base pair amplicon.

7.5.8. Deletion of ADH-A10B

No sequence was identified for a second allele for ADH-A10 in the initial set of 96 sequences but we reasoned that in a diploid organism a second allele existed. At our first attempt we were unable to delete the second allele (ADH-A10B) using the strategy described for ADH-A4B and ADH-B4B. We used the primers A10-IN-F and A10-IN-R to amplify an ˜500 base pair amplicon from genomic DNA from strain DP415 which has the SAT1-flipper inserted into the first ADH-A10 allele, preventing it from amplifying with these primers. The amplicon was cloned and sequenced, the sequence is given as SEQ ID NO: 56.

A10-IN-F: (SEQ ID NO: 121) GAATGGTTCGTGTATGAACTGTGAGTT A10-IN-R: (SEQ ID NO: 122) CCGACTGGTTGATTGCCTTTTCGGAC

We constructed a “pre-targeting” construct comprising two targeting sequences based on SEQ ID NO: 56. A single mutation was introduced into the sequence obtained as SEQ ID NO: 56: a G at position 433 was mutated to a C to destroy an unwanted BsmBI site. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-A10B pre-targeting construct is given as SEQ ID NO: 57. Not shown in SEQ ID NO: 57 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-A10B from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-A10B pre-targeting construct (SEQ ID NO: 57) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-A10B is given as SEQ ID NO: 58. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 base pairs of the genomic sequence of ADH-A10B at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; and between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 58 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 58 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP424 was prepared by integration of the construct shown as SEQ ID NO: 58 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for ADH-A10B. Candida tropicalis strain DP431 was prepared by excision of the targeting construct from the genome of strain DP424, thereby deleting a portion of the coding region of the gene encoding ADH-A10B. Integration and deletion of targeting sequence SEQ ID NO: 58, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were A10-IN-F (SEQ ID NO: 121), A10-IN-R (SEQ ID NO: 122), SAT1-R (SEQ ID NO: 79), and SAT1-F (SEQ ID NO: 80).

Oligonucleotides A10-IN-F and A10-IN-R are designed to anneal to a part of the genome that is missing in strains with deletions in ADH-A10. In such strains they will thus only be able to anneal to and amplify from the second allele ADH-A10B. For strain DP424 (integration of SEQ ID NO: 58), PCR with primers A10-IN-F and SAT1-R produces a 462 base pair amplicon; PCR with primers SAT1-F and A10-IN-R produces a 462 base pair amplicon. PCR from a strain with a wild-type copy of ADH-A10B with primers A10-IN-F and A10-IN-R produces a 488 base pair amplicon. For strain DP431 with a deleted copy of ADH-A10B, PCR with primers A10-IN-F and A10-IN-R produces a 521 base pair amplicon. The amplicons with primers A10-IN-F and A10-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH-A10B, but digestion of the amplicon with either NotI or XhoI will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.

7.5.9. Deletion of ADH-B11B

No sequence was identified for a second allele for ADH-B11 in the initial set of 96 sequences but we reasoned that in a diploid organism a second allele existed. At our first attempt we were unable to delete the second allele (ADH-B11B) using the strategy described for ADH-A4B and ADH-B4B. We used the primers B11-OUT-F and B11-OUT-R to amplify an ˜950 base pair amplicon from genomic DNA from strain DP417 which has the SAT1-flipper inserted into the first ADH-B11 allele, preventing it from amplifying with these primers. The amplicon was cloned and sequenced, the sequence is given as SEQ ID NO: 59.

B11-OUT-F (SEQ ID NO: 121) GAATGGTTCGTGTATGAACTGTGAGTT B11-OUT-R (SEQ ID NO: 122) CCGACTGGTTGATTGCCTTTTCGGAC

We constructed a “pre-targeting” construct comprising two targeting sequences based on SEQ ID NO: 59. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 base pair stuffer fragment and an XhoI restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the ADH-B11B pre-targeting construct is given as SEQ ID NO: 60. Not shown in SEQ ID NO: 60 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli. The sequence was synthesized using standard DNA synthesis techniques well known in the art.

A targeting construct for deletion of ADH-B11B from the Candida tropicalis genome was prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating it into the ADH-B11B pre-targeting construct (SEQ ID NO: 60) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting targeting construct for deletion of ADH-B11B is given as SEQ ID NO: 61. This sequence is a specific example of the construct shown generically in FIG. 4: it has nearly 200 base pair of the genomic sequence of ADH-B11B at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 61 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli. The targeting sequences shown in SEQ ID NO: 61 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.

Candida tropicalis strain DP433 was prepared by integration of the construct shown as SEQ ID NO: 61 into the genome of strain DP431 (Table 3) at the site of the genomic sequence of the gene for ADH-B11B. Candida tropicalis strain DP437 was prepared by excision of the targeting construct from the genome of strain DP433, thereby deleting a portion of the coding region of the gene encoding ADH-B11B. Integration and deletion of targeting sequence SEQ ID NO: 61, and analysis of integrants and excisants were performed as described in Section 7.1.

Sequences of oligonucleotide primers for analysis of strains were:

B11-OUT-F: (SEQ ID NO: 119) B11-IN-R: (SEQ ID NO: 123) CAGACTGGTTGATGGCTTTTTCAGAA SAT1-R: (SEQ ID NO: 79) SAT1-F: (SEQ ID NO: 80)

For strain DP433 (integration of SEQ ID NO: 61), PCR with primers B11-OUT-F and SAT1-R produces a 692 base pair amplicon. PCR from a strain with a wild-type copy of ADH-B11B with primers B11-OUT-F and B11-IN-R produces a 718 base pair amplicon. For strain DP437 with a deleted copy of ADH-B11B, PCR with primers B11-OUT-F and B11-IN-R produces a 751 base pair amplicon. The amplicons with primers B11-OUT-F and B11-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH-B11B, but digestion of the amplicon with either NotI or XhoI will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.

7.6. Insertion of P450 Genes into the Genome of Candida

To achieve novel phenotypes in yeasts of the genus Candida (e.g., Candida tropicalis), including biotransformations of compounds by Candida tropicalis, including chemical conversions not previously obtained, or increased rates of conversion of one or more substrates to one or more products, or increased specificity of conversion of one or more substrates to one or more products, or increased tolerance of a compound by the yeast, or increased uptake of a compound by the yeast, it may be advantageous to incorporate a gene encoding a polypeptide into the genome of the yeast. Expression of the polypeptide in the yeast then allows the phenotype of the yeast to be modified.

In some embodiments of the invention it may be advantageous to integrate a gene encoding a polypeptide into a strain of Candida tropicalis in which one or more of the alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B1B and ADH-B11 have been disrupted. In some embodiments of the invention it may be advantageous to integrate a gene encoding a polypeptide into a yeast strain of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides within the coding region, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence or at least 75% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 70% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 65% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 60% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence with one of the Candida tropicalis genes ADH-A4 (SEQ ID NO: 39), ADH-B4 (SEQ ID NO: 42), ADH-A10 (SEQ ID NO: 40), ADH-A10B (SEQ ID NO: 56), ADH-B11 (SEQ ID NO: 43).

In some embodiments it may be advantageous to integrate a gene encoding a polypeptide into a yeast strain of the genus Candida in which (i) one or more alcohol dehydrogenase genes have been disrupted and (ii) the disrupted alcohol dehydrogenase comprises a first peptide. In some embodiments said first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments said first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a second peptide. In some embodiments said second peptide has the sequence QYATADAVQAA (SEQ ID NO: 158). In some embodiments said second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164). In some embodiments said second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165). In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a third peptide. In some embodiments said third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments said third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide. In some embodiments said fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160). In some embodiments said fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments said fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments said fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169). In some embodiments said fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG. (SEQ ID NO: 170). In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide. In some embodiments said fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).

Cytochrome P450s are of particular utility in the hydroxylation of a variety of substrates including fatty acids. Different cytochrome P450s are known to have different substrate and regiospecificities and different specific activities. It is therefore useful in some embodiments of the invention to incorporate a gene encoding a cytochrome P450 into the genome of the yeast. The exact P450 to be used will depend upon the substrate and the position on the substrate to be hydroxylated. A list of P450 enzymes that may be of utility in the hydroxylation of substrates when expressed within a yeast cell are given in Table 4.

TABLE 4 First Database Accession Second Database Number Accession Number Name Species gi 29469875 gb AAO73958.1 CYP52A17 Candida tropicalis gi 29469877 gb AAO73959.1 CYP52A18 Candida tropicalis gi 231889 sp P30610.1 CP52H_CANTR (Cytochrome P450 52A8) gi 3913326 sp Q12586.1 CP52I_CANMA (Cytochrome P450 52A9) gi 29469881 gb AAO73961.1 CYP52A20 Candida tropicalis gi 29469879 gb AAO73960.1 CYP52A19 Candida tropicalis gi 3913329 sp Q12589.1 CP52K_CANMA (Cytochrome P450 52A11) gi 3913328 sp Q12588.1 CP52J_CANMA (Cytochrome P450 52A10) gi 68492087 ref XP_710174.1 P450 drug resistance protein Candida albicans gi 3395458 emb CAA75058.1 alk8 Candida albicans gi 68474594 ref XP_718670.1 CaO19.7513 Candida albicans gi 29469865 gb AAO73953.1 CYP52A13 Candida tropicalis gi 149239010 ref XP_001525381.1 cytochrome P450 52A11 Lodderomyces elongisporus gi 29469867 gb AAO73954.1 CYP52A14 Candida tropicalis gi 7548332 gb AAA34353.2 cytochrome P-450-alk2 Candida tropicalis gi 732622 emb CAA39366.1 n-alkane inducible Candida maltosa cytochrome P-450 gi 231886 sp P30607.1 CP52B_CANTR (Cytochrome P450 52A2) gi 68474592 ref XP_718669.1 CaO19.7512 Candida albicans gi 150864612 ref XP_001383506.2 n-alkane inducible Pichia stipitis cytochrome P-450 gi 231888 sp P30609.1 CP52G_CANTR (Cytochrome P450 52A7) gi 298217 gb AAB24479.1 cytochrome P450 Candida tropicalis monoxygenase alk4, P450 alk4 = CYP52A7 gene product {alkane-inducible} gi 149246109 ref XP_001527524.1 cytochrome P450 52A2 Lodderomyces elongisporus gi 29469869 gb AAO73955.1 CYP52A15 Candida tropicalis gi 190319368 gb AAD22536.2 AF103948_1 cytochrome Debaryomyces hansenii P450 alkane hydroxylase gi 146419207 ref XP_001485567.1 cytochrome P450 52A12 Pichia guilliermondii gi 29469863 gb AAO73952.1 CYP52A12 Candida tropicalis gi 50423067 ref XP_460112.1 DEHA0E19635g Debaryomyces hansenii gi 29469871 gb AAO73956.1 CYP52A16 Candida tropicalis gi 199432969 emb CAG88381.2 DEHA2E18612p Debaryomyces hansenii gi 170892 gb AAA34354.1 cytochrome P-450-alk1 Candida tropicalis gi 50423065 ref XP_460111.1 DEHA0E19613g Debaryomyces hansenii gi 1169075 sp P10615.3 CP52A_CANTR (Cytochrome P450 52A1) gi 226487 prf 1515252A cytochrome P450alk1 gi 732623 emb CAA39367.1 n-alkane inducible Candida maltosa cytochrome P-450 gi 146413358 ref XP_001482650.1 PGUG_05670 Pichia guilliermondii gi 117182 sp P16141.3 CP52D_CANMA (Cytochrome P450 52A4) gi 2608 emb CAA36197.1 unnamed protein product Candida maltosa gi 231887 sp P30608.1 CP52F_CANTR (Cytochrome P450 52A6) gi 199432970 emb CAG88382.2 DEHA2E18634p Debaryomyces hansenii gi 190349008 gb EDK41572.2 PGUG_05670 Pichia guilliermondii gi 150864699 ref XP_001383636.2 Cytochrome P450 52A12 Pichia stipitis (Alkane hydroxylase 1) (Alkane-inducible p450alk 1) (DH-ALK2) gi 117181 sp P16496.3 CP52C_CANMA (Cytochrome P450 52A3) gi 199432968 emb CAG88380.2 DEHA2E18590p Debaryomyces hansenii gi 50423063 ref XP_460110.1 DEHA0E19591g Debaryomyces hansenii gi 553118 gb AAA34320.1 alkane hydroxylating cytochrome P-450 gi 117183 sp P24458.1 CP52E_CANMA (Cytochrome P450 52A5) gi 68475852 ref XP_717999.1 potential alkane Candida albicans hydroxylating monooxygenase P450 gi 18203639 sp Q9Y758.1 CP52M_DEBHA (Cytochrome P450 52A13) gi 146412241 ref XP_001482092.1 cytochrome P450 52A13 Pichia guilliermondii gi 126134585 ref XP_001383817.1 Cytochrome P450 52A13 Pichia stipitis (Alkane hydroxylase 2) (Alkane-inducible p450alk2) (DH-ALK2) gi 50418551 ref XP_457792.1 DEHA0C02981g Debaryomyces hansenii gi 149236533 ref XP_001524144.1 cytochrome P450 52A5 Lodderomyces elongisporus gi 150864746 ref XP_001383710.2 Cytochrome P450 52A6 Pichia stipitis (CYPLIIA6) (Alkane- inducible P450-ALK3) gi 149239404 ref XP_001525578.1 cytochrome P450 52A3 Lodderomyces elongisporus gi 50417817 ref XP_457727.1 DEHA0C01177g Debaryomyces hansenii gi 199430432 emb CAG85755.2 DEHA2C01100p Debaryomyces hansenii gi 149239402 ref XP_001525577.1 cytochrome P450 52A8 Lodderomyces elongisporus gi 29469873 gb AAO73957.1 CYP52D2 Candida tropicalis gi 150866745 ref XP_001386440.2 Cytochrome P450 52A3 Pichia stipitis (CYPLIIA3) (Alkane- inducible P450-ALK1-A) (P450-CM1) (CYP52A3-A) (Cytochrome P-450ALK) gi 190347603 gb EDK39907.2 PGUG_04005 Pichia guilliermondii gi 146414612 ref XP_001483276.1 PGUG_04005 Pichia guilliermondii gi 3913325 sp Q12585.1 CP52T_CANMA (Cytochrome P450 52D1) gi 50553995 ref XP_504406.1 YALI0E25982p Yarrowia lipolytica gi 3298289 dbj BAA31433.1 ALK1 Yarrowia lipolytica gi 50554897 ref XP_504857.1 YALI0F01320p Yarrowia lipolytica gi 50545727 ref XP_500402.1 YALI0B01848p Yarrowia lipolytica gi 50546066 ref XP_500560.1 YALI0B06248p Yarrowia lipolytica gi 50547357 ref XP_501148.1 YALI0B20702p Yarrowia lipolytica gi 50546771 ref XP_500855.1 YALI0B13816p Yarrowia lipolytica gi 50546773 ref XP_500856.1 YALI0B13838p Yarrowia lipolytica gi 70982077 ref XP_746567.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 119487140 ref XP_001262425.1 cytochrome P450 alkane Neosartorya fischeri hydroxylase gi 50545119 ref XP_500097.1 YALI0A15488p Yarrowia lipolytica gi 115387741 ref XP_001211376.1 cytochrome P450 52A12 Aspergillus terreus gi 145248800 ref XP_001400739.1 An14g01110 Aspergillus niger gi 121714465 ref XP_001274843.1 cytochrome P450 alkane Aspergillus clavatus hydroxylase gi 50545471 ref XP_500273.1 YALI0A20130p Yarrowia lipolytica gi 212541280 ref XP_002150795.1 cytochrome P450 alkane Penicillium marneffei hydroxylase gi 169783066 ref XP_001825995.1 Aspergillus oryzae gi 67541935 ref XP_664735.1 AN7131.2 Aspergillus nidulans gi 218716670 gb EED16091.1 cytochrome P450 alkane Talaromyces stipitatus hydroxylase gi 211584648 emb CAP74173.1 Pc14g00320 Penicillium chrysogenum gi 68475719 ref XP_718066.1 potential alkane Candida albicans hydroxylating monooxygenase P450 fragment gi 231890 sp P30611.1 CP52N_CANTR (Cytochrome P450 52B1) gi 50553800 ref XP_504311.1 YALI0E23474p Yarrowia lipolytica gi 115391153 ref XP_001213081.1 ATEG_03903 Aspergillus terreus gi 1169076 sp P43083.1 CP52V_CANAP (Cytochrome P450 52E1) gi 212537573 ref XP_002148942.1 cytochrome P450 family Penicillium marneffei protein gi 119480837 ref XP_001260447.1 cytochrome P450 family Neosartorya fischeri protein gi 159129370 gb EDP54484.1 cytochrome P450 family Aspergillus fumigatus protein gi 71001214 ref XP_755288.1 cytochrome P450 family Aspergillus fumigatus protein gi 50548557 ref XP_501748.1 YALI0C12122p Yarrowia lipolytica gi 211592844 emb CAP99212.1 Pc22g19240 Penicillium chrysogenum gi 231891 sp P30612.1 CP52P_CANTR (Cytochrome P450 52C1) gi 3913327 sp Q12587.1 CP52Q_CANMA (Cytochrome P450 52C2) gi 50548395 ref XP_501667.1 YALI0C10054p Yarrowia lipolytica gi 145248373 ref XP_001396435.1 An13g03000 Aspergillus niger gi 169783674 ref XP_001826299.1 Aspergillus oryzae gi 169774249 ref XP_001821592.1 Aspergillus oryzae gi 212536398 ref XP_002148355.1 cytochrome P450 alkane Penicillium marneffei hydroxylase gi 211590140 emb CAP96310.1 Pc21g14130 Penicillium chrysogenum gi 189200681 ref XP_001936677.1 cytochrome P450 52A12 Pyrenophora tritici- repentis gi 121698992 ref XP_001267871.1 cytochrome P450 family Aspergillus clavatus protein gi 154310961 ref XP_001554811.1 BC1G_06459 Botryotinia fuckeliana gi 119497443 ref XP_001265480.1 cytochrome P450 alkane Neosartorya fischeri hydroxylase gi 67539774 ref XP_663661.1 AN6057.2 Aspergillus nidulans gi 3913324 sp Q12573.1 CP52W_CANAP (Cytochrome P450 52E2) gi 159130401 gb EDP55514.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 70990140 ref XP_749919.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 212543867 ref XP_002152088.1 N-alkane-inducible Penicillium marneffei cytochrome P450 ATCC 18224 gi 189204508 ref XP_001938589.1 cytochrome P450 52A12 Pyrenophora tritici- repentis gi 67904794 ref XP_682653.1 AN9384.2 Aspergillus nidulans gi 115401146 ref XP_001216161.1 ATEG_07540 Aspergillus terreus gi 169765686 ref XP_001817314.1 Aspergillus oryzae gi 156034334 ref XP_001585586.1 SS1G_13470 Sclerotinia sclerotiorum gi 115389132 ref XP_001212071.1 ATEG_02893 Aspergillus terreus gi 149249004 ref XP_001528842.1 LELG_05768 Lodderomyces elongisporus gi 119490743 ref XP_001263094.1 n-alkane-inducible Neosartorya fischeri cytochrome P450 gi 169598696 ref XP_001792771.1 SNOG_02153 Phaeosphaeria nodorum gi 145233653 ref XP_001400199.1 An02g10700 Aspergillus niger gi 121703415 ref XP_001269972.1 cytochrome P450 alkane Aspergillus clavatus hydroxylase gi 145244813 ref XP_001394678.1 An11g07010 Aspergillus niger gi 115400535 ref XP_001215856.1 ATEG_06678 Aspergillus terreus gi 156054264 ref XP_001593058.1 SS1G_05980 Sclerotinia sclerotiorum gi 145235009 ref XP_001390153.1 An03g02570 Aspergillus niger gi 121714697 ref XP_001274959.1 n-alkane-inducible Aspergillus clavatus cytochrome P450 gi 115383936 ref XP_001208515.1 ATEG_01150 Aspergillus terreus gi 119188703 ref XP_001244958.1 CIMG_04399 Coccidioides immitis gi 154303347 ref XP_001552081.1 BC1G_09422 Botryotinia fuckeliana gi 68469246 ref XP_721410.1 potential n-alkane inducible Candida albicans cytochrome P-450 gi 211588353 emb CAP86458.1 Pc20g11290 Penicillium chrysogenum gi 218719422 gb EED18842.1 cytochrome P450 Talaromyces stipitatus gi 189196472 ref XP_001934574.1 cytochrome P450 52A11 Pyrenophora tritici- repentis gi 145228377 ref XP_001388497.1 An01g00510 Aspergillus niger gi 145243810 ref XP_001394417.1 An11g04220 Aspergillus niger gi 119467390 ref XP_001257501.1 n-alkane-inducible Neosartorya fischeri cytochrome P450 gi 218713692 gb EED13116.1 cytochrome P450 alkane Talaromyces stipitatus hydroxylase gi 156040904 ref XP_001587438.1 SS1G_11430 Sclerotinia sclerotiorum gi 211588608 emb CAP86724.1 Pc20g13950 Penicillium chrysogenum gi 189210960 ref XP_001941811.1 cytochrome P450 52A11 Pyrenophora tritici- repentis gi 154300280 ref XP_001550556.1 BC1G_11329 Botryotinia fuckeliana gi 39965179 ref XP_365075.1 MGG_09920 Magnaporthe grisea gi 70984521 ref XP_747767.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 164424932 ref XP_958030.2 NCU09115 Neurospora crassa gi 169785321 ref XP_001827121.1 Aspergillus oryzae gi 171687345 ref XP_001908613.1 Podospora anserina gi 495225 dbj BAA05145.1 n-alkane-inducible Candida maltosa cytochrome P-450 gi 169778468 ref XP_001823699.1 Aspergillus oryzae gi 685237 emb CAA35593.1 cytochrome P-450-alk2 Candida tropicalis gi 115398792 ref XP_001214985.1 ATEG_05807 Aspergillus terreus gi 156045685 ref XP_001589398.1 SS1G_10037 Sclerotinia sclerotiorum gi 116181964 ref XP_001220831.1 CHGG_01610 Chaetomium globosum gi 212539338 ref XP_002149824.1 N-alkane-inducible Penicillium marneffei cytochrome P450 gi 55823915 gb AAV66104.1 cytochrome P450 Fusarium heterosporum gi 169786131 ref XP_001827526.1 Aspergillus oryzae gi 67526919 ref XP_661521.1 AN3917.2 Aspergillus nidulans gi 57157397 dbj BAD83681.1 cytochrome P-450 Alternaria solani gi 39954838 ref XP_364111.1 MGG_08956 Magnaporthe grisea gi 46108804 ref XP_381460.1 FG01284.1 Gibberella zeae gi 167962420 dbj BAG09241.1 n-alkane inducible Candida maltosa cytochrome P-450 gi 119469615 ref XP_001257962.1 cytochrome P450 alkane Neosartorya fischeri hydroxylase gi 70991773 ref XP_750735.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 171679185 ref XP_001904540.1 unnamed protein product Podospora anserina gi 119488606 ref XP_001262753.1 n-alkane-inducible Neosartorya fischeri cytochrome P450 gi 218722969 gb EED22387.1 cytochrome P450 Talaromyces stipitatus gi 145243244 ref XP_001394159.1 An11g01550 Aspergillus niger gi 212533853 ref XP_002147083.1 N-alkane-inducible Penicillium marneffei cytochrome P450 gi 218720976 gb EED20395.1 cytochrome P450 alkane Talaromyces stipitatus hydroxylase gi 145604320 ref XP_362943.2 MGG_08494 Magnaporthe grisea gi 154319876 ref XP_001559255.1 BC1G_02419 Botryotinia fuckeliana gi 154272319 ref XP_001537012.1 HCAG_08121 Ajellomyces capsulatus gi 39976331 ref XP_369556.1 MGG_05908 Magnaporthe grisea gi 116200125 ref XP_001225874.1 CHGG_08218 Chaetomium globosum gi 218722681 gb EED22099.1 cytochrome P450 alkane Talaromyces stipitatus hydroxylase gi 145606889 ref XP_361347.2 MGG_03821 Magnaporthe grisea gi 211592275 emb CAP98620.1 Pc22g13320 Penicillium chrysogenum gi 171688034 ref XP_001908957.1 unnamed protein product Podospora anserina gi 211587061 emb CAP94723.1 Pc18g04990 Penicillium chrysogenum gi 169612986 ref XP_001799910.1 SNOG_09621 Phaeosphaeria nodorum gi 212539354 ref XP_002149832.1 N-alkane-inducible Penicillium marneffei cytochrome P450 gi 212533239 ref XP_002146776.1 cytochrome P450 alkane Penicillium marneffei hydroxylase gi 41079162 gb AAR99474.1 alkane monooxygenase P- Graphium sp. 450 gi 159122944 gb EDP48064.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 67537376 ref XP_662462.1 AN4858.2 Aspergillus nidulans gi 39954738 ref XP_364102.1 MGG_08947 Magnaporthe grisea gi 39968921 ref XP_365851.1 MGG_10071 Magnaporthe grisea gi 70983886 ref XP_747469.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 171691438 ref XP_001910644.1 unnamed protein product Podospora anserina gi 119193452 ref XP_001247332.1 CIMG_01103 Coccidioides immitis gi 10303293 emb CAC10088.1 related to n-alkane-inducible Neurospora crassa cytochrome P450 gi 169626152 ref XP_001806478.1 SNOG_16355 Phaeosphaeria nodorum gi 119191908 ref XP_001246560.1 CIMG_00331 Coccidioides immitis gi 154296077 ref XP_001548471.1 BC1G_12768 Botryotinia fuckeliana gi 164429645 ref XP_964653.2 NCU02031 Neurospora crassa gi 12311700 emb CAC24473.1 Candida albicans gi 154305169 ref XP_001552987.1 BC1G_08879 Botryotinia fuckeliana gi 39978177 ref XP_370476.1 MGG_06973 Magnaporthe grisea gi 70982576 ref XP_746816.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 154319145 ref XP_001558890.1 BC1G_02524 Botryotinia fuckeliana gi 46127885 ref XP_388496.1 FG08320.1 Gibberella zeae gi 32330665 gb AAP79879.1 cytochrome P450 Phanerochaete monooxygenase pc-3 chrysosporium gi 116193605 ref XP_001222615.1 CHGG_06520 Chaetomium globosum gi 145241598 ref XP_001393445.1 An09g01270 Aspergillus niger gi 149210127 ref XP_001522438.1 MGCH7_ch7g545 Magnaporthe grisea gi 121699244 ref XP_001267956.1 cytochrome P450 alkane Aspergillus clavatus hydroxylase gi 156032429 ref XP_001585052.1 SS1G_13912 Sclerotinia sclerotiorum gi 159122551 gb EDP47672.1 cytochrome P450 alkane Aspergillus fumigatus hydroxylase gi 145613078 ref XP_001412594.1 MGG_12496 Magnaporthe grisea gi 212531571 ref XP_002145942.1 N-alkane-inducible Penicillium marneffei cytochrome P450 gi 145252862 ref XP_001397944.1 An16g06420 Aspergillus niger gi 169855683 ref XP_001834508.1 CC1G_02244 Coprinopsis cinerea okayama gi 212530338 ref XP_002145326.1 N-alkane-inducible Penicillium marneffei cytochrome P450 gi 61657996 gb AAX49400.1 cytochrome P450 Phanerochaete monooxygenase pc-2 chrysosporium gi 170110164 ref XP_001886288.1 CYP63 cytochrome P450 Laccaria bicolor monooxygenase-like protein gi 146323950 ref XP_748328.2 cytochrome P450 Aspergillus fumigatus oxidoreductase/alkane hydroxylase gi 156042346 ref XP_001587730.1 SS1G_10970 Sclerotinia sclerotiorum gi 189196282 ref XP_001934479.1 cytochrome P450 71A23 Pyrenophora tritici- repentis gi 18369901 gb AAL67906.1 cytochrome P450 Phanerochaete monooxygenase pc-2 chrysosporium gi 218714942 gb EED14365.1 cytochrome P450 Talaromyces stipitatus gi 170106497 ref XP_001884460.1 cytochrome P450 Laccaria bicolor gi 169865534 ref XP_001839366.1 CC1G_08233 Coprinopsis cinerea okayama gi 169855669 ref XP_001834501.1 CC1G_02237 Coprinopsis cinerea okayama gi 189197495 ref XP_001935085.1 cytochrome P450 52A1 Pyrenophora tritici- repentis gi 218713646 gb EED13070.1 cytochrome P450 Talaromyces stipitatus gi 170106217 ref XP_001884320.1 cytochrome P450 Laccaria bicolor gi 116197088 ref XP_001224356.1 CHGG_05142 Chaetomium globosum gi 18369899 gb AAL67905.1 cytochrome P450 Phanerochaete monooxygenase pc-1 chrysosporium gi 154312290 ref XP_001555473.1 BC1G_06178 Botryotinia fuckeliana gi 156064223 ref XP_001598033.1 SS1G_00119 Sclerotinia sclerotiorum gi 156039263 ref XP_001586739.1 SS1G_11768 Sclerotinia sclerotiorum gi 170105206 ref XP_001883816.1 Laccaria bicolor gi 169613228 ref XP_001800031.1 SNOG_09744 Phaeosphaeria nodorum gi 169863123 ref XP_001838184.1 CC1G_12233 Coprinopsis cinerea okayama gi 67902848 ref XP_681680.1 AN8411.2 Aspergillus nidulans gi 158392452 emb CAO91865.1 monooxygenase Penicillium expansum gi 169857173 ref XP_001835239.1 CC1G_07782 Coprinopsis cinerea okayama gi 169781220 ref XP_001825073.1 Aspergillus oryzae gi 67540302 ref XP_663925.1 AN6321.2 Aspergillus nidulans gi 145234553 ref XP_001389925.1 An03g00180 Aspergillus niger gi 170106275 ref XP_001884349.1 Laccaria bicolor gi 145610012 ref XP_366716.2 MGG_02792 Magnaporthe grisea gi 119473653 ref XP_001258702.1 cytochrome P450 Neosartorya fischeri monooxygenase gi 118026355 emb CAL69594.1 Cordyceps bassiana gi 154309945 ref XP_001554305.1 BC1G_06893 Botryotinia fuckeliana gi 211593324 emb CAP99706.1 Pc22g24180 Penicillium chrysogenum gi 170111410 ref XP_001886909.1 cytochrome P450 Laccaria bicolor monooxygenase CYP63 gi 169864610 ref XP_001838912.1 CC1G_05465 Coprinopsis cinerea okayama gi 145240007 ref XP_001392650.1 An08g05330 Aspergillus niger gi 115433302 ref XP_001216788.1 Aspergillus terreus gi 121701751 ref XP_001269140.1 Cytochrome P450 Aspergillus clavatus oxidoreductase gi 154289956 ref XP_001545581.1 BC1G_15919 Botryotinia fuckeliana gi 212527006 ref XP_002143660.1 cytochrome P450 alkane Penicillium marneffei hydroxylase gi 156054506 ref XP_001593179.1 SS1G_06101 Sclerotinia sclerotiorum gi 167962125 dbj BAG09240.1 n-alkane inducible Candida maltosa cytochrome P-450 gi 169610561 ref XP_001798699.1 SNOG_08385 Phaeosphaeria nodorum gi 154322320 ref XP_001560475.1 BC1G_01307 Botryotinia fuckeliana gi 171986596 gb ACB59278.1 cytochrome P450 Pseudozyma flocculosa monooxygenase gi 169850022 ref XP_001831709.1 CC1G_12229 Coprinopsis cinerea okayama gi 84514171 gb ABC59094.1 cytochrome P450 Medicago truncatula monooxygenase CYP704G9 gi 157349259 emb CAO24405.1 Vitis vinifera gi 154322983 ref XP_001560806.1 BC1G_00834 Botryotinia fuckeliana gi 71726950 gb AAZ39646.1 cytochrome P450 Petunia x hybrida monooxygenase gi 2160323 dbj BAA05146.1 n-alkane-inducible Candida maltosa cytochrome P-450 gi 218717320 gb EED16741.1 cytochrome P450 Talaromyces stipitatus gi 118485860 gb ABK94777.1 Populus trichocarpa gi 71024781 ref XP_762620.1 UM06473.1 Ustilago maydis gi 58265104 ref XP_569708.1 Cryptococcus neoformans var. neoformans gi 169596949 ref XP_001791898.1 SNOG_01251 Phaeosphaeria nodorum gi 157355912 emb CAO49769.1 Vitis vinifera gi 134109309 ref XP_776769.1 CNBC2600 Cryptococcus neoformans var. neoformans gi 157349262 emb CAO24408.1 Vitis vinifera gi 147765747 emb CAN60189.1 Vitis vinifera gi 169864676 ref XP_001838945.1 CC1G_05498 Coprinopsis cinerea okayama gi 157352095 emb CAO43102.1 Vitis vinifera gi 147791153 emb CAN63571.1 Vitis vinifera gi 84514173 gb ABC59095.1 cytochrome P450 Medicago truncatula monooxygenase CYP704G7 gi 71024761 ref XP_762610.1 UM06463.1 Ustilago maydis gi 157355911 emb CAO49768.1 Vitis vinifera gi 115451645 ref NP_001049423.1 Os03g0223100 Oryza sativa gi 22748335 gb AAN05337.1 cytochrome P450 Oryza sativa gi 168059245 ref XP_001781614.1 Physcomitrella patens subsp. patens gi 15225499 ref NP_182075.1 CYP704A2 (cytochrome Arabidopsis thaliana P450, family 704, subfamily A, polypeptide 2) oxygen binding gi 75319885 sp Q50EK3.1 C04C1_PINTA (Cytochrome P450 704C1) gi 167521978 ref XP_001745327.1 Monosiga brevicollis gi 21536522 gb AAM60854.1 cytochrome P450-like Arabidopsis thaliana protein gi 15242759 ref NP_201150.1 CYP94B1 (cytochrome Arabidopsis thaliana P450, family 94, subfamily B, polypeptide 1) oxygen binding gi 168031659 ref XP_001768338.1 Physcomitrella patens subsp.patens gi 157339131 emb CAO42482.1 Vitis vinifera gi 30682301 ref NP_196442.2 cytochrome P450 family Arabidopsis thaliana protein gi 8346562 emb CAB93726.1 cytochrome P450-like Arabidopsis thaliana protein gi 2344895 gb AAC31835.1 cytochrome P450 Arabidopsis thaliana gi 30689861 ref NP_850427.1 CYP704A1 (cytochrome Arabidopsis thaliana P450, family 704, subfamily A, polypeptide 1) oxygen binding gi 15221776 ref NP_173862.1 CYP86C1 (cytochrome Arabidopsis thaliana P450, family 86, subfamily C, polypeptide 1) oxygen binding gi 147793015 emb CAN77648.1 Vitis vinifera gi 157356646 emb CAO62841.1 Vitis vinifera gi 147844260 emb CAN80040.1 Vitis vinifera gi 215466577 gb EEB96517.1 MPER_04337 Moniliophthora perniciosa gi 15222515 ref NP_176558.1 CYP86A7 (cytochrome Arabidopsis thaliana P450, family 86, subfamily A, polypeptide 7) oxygen binding gi 194697724 gb ACF82946.1 Zea mays gi 168021353 ref XP_001763206.1 Physcomitrella patens subsp. patens gi 115483036 ref NP_001065111.1 Os10g0525000 Oryza sativa (japonica cultivar-group) gi 157338660 emb CAO42011.1 Vitis vinifera gi 147836212 emb CAN75428.1 Vitis vinifera gi 5042165 emb CAB44684.1 cytochrome P450-like Arabidopsis thaliana protein gi 79326551 ref NP_001031814.1 CYP96A10 (cytochrome Arabidopsis thaliana P450, family 96, subfamily A, polypeptide 10) heme binding/iron ion binding/ monooxygenase gi 26452145 dbj BAC43161.1 cytochrome P450 Arabidopsis thaliana gi 110289450 gb AAP54707.2 Cytochrome P450 family Oryza sativa protein, expressed gi 21593258 gb AAM65207.1 cytochrome P450 Arabidopsis thaliana gi 115483034 ref NP_001065110.1 Os10g0524700 Oryza sativa gi 118486379 gb ABK95030.1 Populus trichocarpa gi 10442763 gb AAG17470.1 AF123610_9 cytochrome Triticum aestivum P450 gi 125532704 gb EAY79269.1 OsI_34384 Oryza sativa gi 15237250 ref NP_197710.1 CYP86B1 (cytochrome Arabidopsis thaliana P450, family 86, subfamily B, polypeptide 1) oxygen binding gi 125549414 gb EAY95236.1 OsI_17053 Oryza sativa gi 110289453 gb AAP54710.2 Cytochrome P450 family Oryza sativa protein gi 20146744 gb AAM12480.1 AC074232_7 cytochrome Oryza sativa P450-like protein gi 218184911 gb EEC67338.1 OsI_34388 Oryza sativa Indica Group gi 125549325 gb EAY95147.1 OsI_16965 Oryza sativa Indica Group gi 198472816 ref XP_002133118.1 GA29000 Drosophila pseudoobscura pseudoobscura gi 195574346 ref XP_002105150.1 GD21336 Drosophila simulans gi 168024173 ref XP_001764611.1 Physcomitrella patens subsp. patens gi 115440549 ref NP_001044554.1 Os01g0804400 Oryza sativa (japonica cultivar-group) gi 15223657 ref NP_176086.1 CYP96A15/MAH1 (MID- Arabidopsis thaliana CHAIN ALKANE HYDROXYLASE 1) oxygen binding gi 125540131 gb EAY86526.1 OsI_07906 Oryza sativa gi 115460030 ref NP_001053615.1 Os04g0573900 Oryza sativa (japonica cultivar-group) gi 157349258 emb CAO24404.1 Vitis vinifera gi 157346575 emb CAO16644.1 Vitis vinifera gi 147835182 emb CAN76753.1 Vitis vinifera gi 195613956 gb ACG28808.1 Zea mays gi 194753285 ref XP_001958947.1 GF12635 Drosophila ananassae gi 156546811 ref XP_001606040.1 Nasonia vitripennis gi 125583181 gb EAZ24112.1 OsJ_007595 Oryza sativa (japonica cultivar-group) gi 15229477 ref NP_189243.1 CYP86C2 (cytochrome Arabidopsis thaliana P450, family 86, subfamily C, polypeptide 2) oxygen binding gi 940446 emb CAA62082.1 cytochrome p450 Arabidopsis thaliana gi 115447789 ref NP_001047674.1 Os02g0666500 Oryza sativa (japonica cultivar-group) gi 15227788 ref NP_179899.1 CYP96A1 (cytochrome Arabidopsis thaliana P450, family 96, subfamily A, polypeptide 1) oxygen binding gi 195503768 ref XP_002098791.1 GE23738 Drosophila yakuba gi 147804860 emb CAN66874.1 Vitis vinifera gi 84514169 gb ABC59093.1 cytochrome P450 Medicago truncatula monooxygenase CYP94C9 gi 19698839 gb AAL91155.1 cytochrome P450 Arabidopsis thaliana gi 15237768 ref NP_200694.1 CYP86A1 (cytochrome Arabidopsis thaliana P450, family 86, subfamily A, polypeptide 1) oxygen binding gi 157353969 emb CAO46510.1 Vitis vinifera gi 169865676 ref XP_001839436.1 CC1G_06649 Coprinopsis cinerea okayama gi 85001697 gb ABC68403.1 cytochrome P450 Glycine max monooxygenase CYP86A24 gi 115466172 ref NP_001056685.1 Os06g0129900 Oryza sativa gi 195637782 gb ACG38359.1 cytochrome P450 86A2 Zea mays gi 194704220 gb ACF86194.1 Zea mays gi 71006408 ref XP_757870.1 UM01723.1 Ustilago maydis 521 gi 195161677 ref XP_002021689.1 GL26642 Drosophila persimilis gi 115459886 ref NP_001053543.1 Os04g0560100 Oryza sativa gi 194704096 gb ACF86132.1 Zea mays gi 147773635 emb CAN67559.1 Vitis vinifera gi 125575195 gb EAZ16479.1 OsJ_030688 Oryza sativa gi 115482616 ref NP_001064901.1 Os10g0486100 Oryza sativa gi 71726942 gb AAZ39642.1 cytochrome P450 fatty acid Petunia x hybrida omega-hydroxylase gi 195626182 gb ACG34921.1 cytochrome P450 86A1 Zea mays gi 194907382 ref XP_001981543.1 GG11553 Drosophila erecta gi 71006688 ref XP_758010.1 UM01863.1 Ustilago maydis gi 157346247 emb CAO15944.1 Vitis vinifera gi 116830948 gb ABK28430.1 Arabidopsis thaliana gi 13641298 gb AAK31592.1 cytochrome P450 Brassica rapa subsp. pekinensis gi 2258321 gb AAB63277.1 cytochrome P450 Phanerochaete chrysosporium gi 15218671 ref NP_174713.1 CYP94D1 (cytochrome Arabidopsis thaliana P450, family 94, subfamily D, polypeptide 1) oxygen binding gi 195623910 gb ACG33785.1 cytochrome P450 86A1 Zea mays gi 157337152 emb CAO21498.1 Vitis vinifera binding gi 195623910 gb ACG33785.1 cytochrome P450 86A1 Zea mays gi 157337152 emb CAO21498.1 Vitis vinifera

In some embodiments of the invention it is advantageous to integrate one or more genes encoding a P450 enzyme-into a yeast strain, a species of the genus Candida or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of the substrate have been disrupted. In some embodiments, a strain of yeast in which one or more cytochrome P450s or one or more alcohol oxidase or one or more alcohol dehydrogenase have been disrupted will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.

In some embodiments of the invention it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B have been disrupted. In some embodiments of the invention it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which at least one of the fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B have been disrupted. In some embodiments of the invention it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 have been disrupted. In some embodiments of the invention it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which one or more of the alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B1B and ADH-B11 have been disrupted. In some embodiments of the invention it may be advantageous to integrate a gene encoding a cytochrome P450 into a yeast species of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 75% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 70% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 65% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 60% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence within the coding region with one of the Candida tropicalis genes ADH-A4 (SEQ ID NO: 39), ADH-B4 (SEQ ID NO: 42), ADH-A10 (SEQ ID NO: 40), ADH-A10B (SEQ ID NO: 56), ADH-B11 (SEQ ID NO: 43).

In some embodiments it may be advantageous to integrate a gene encoding a cytochrome P450 into a yeast strain of the genus Candida in which (i) one or more alcohol dehydrogenase genes have been disrupted and (ii) the disrupted alcohol dehydrogenase comprises a first peptide. In some embodiments the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163). In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a second peptide. In some embodiments the second peptide has the sequence QYATADAVQAA (SEQ ID NO: 158). In some embodiments the second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164). In some embodiments the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a third peptide. In some embodiments the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide. In some embodiments the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide. In some embodiments said fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).

In some embodiments of the invention it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted. In some embodiments it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which one or more of the cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted. In some embodiments it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B, alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the β-oxidation pathway has also been disrupted.

In some embodiments, a cytochrome P450 is integrated into a strain of Candida tropicalis in which endogenous cyocrhrome P450s have been disrupted.

In some embodiments, a cytochrome P450 is integrated into a strain of Candida in which endogenous cyocrhrome P450s have been disrupted.

In some embodiments, a cytochrome P450 is integrated into a strain of yeast of a species of the genus Candida in which endogenous cyocrhrome P450s have been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a yeast strain, a species of Candida, or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of a fatty acid substrate (e.g., a α-carboxyl-ω-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an α,ω-dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof) have been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, or one or more disrupted alcohol oxidases, or one or more disrupted alcohol dehydrogenases present in the strain of yeast will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, one or more disrupted alcohol oxidases, and one or more disrupted alcohol dehydrogenases will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B have been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which endogenous alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 have been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which endogenous cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B, alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the β-oxidation pathway has also been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which endogenous cytochrome P450s have been disrupted.

In some embodiments, one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida in which endogenous cytochrome P450s have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a yeast strain, a species of Candida, or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of a fatty acid substrate (e.g., a α-carboxyl-ω-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an α,ω-dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof) have been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, or one or more disrupted alcohol oxidases, or one or more disrupted alcohol dehydrogenases present in the strain of yeast will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, one or more disrupted alcohol oxidases, and one or more disrupted alcohol dehydrogenases will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of yeast species of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein at least one disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides within the coding region, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 75% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 70% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 65% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 60% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence with one of the Candida tropicalis genes ADH-A4 (SEQ ID NO: 39), ADH-B4 (SEQ ID NO: 42), ADH-A10 (SEQ ID NO: 40), ADH-A10B (SEQ ID NO: 56), ADH-B11 (SEQ ID NO: 43).

In some embodiments, a gene listed in Table 4 is integrated into a strain of yeast of the genus Candida in which (i) one or more alcohol dehydrogenase genes has been disrupted and (ii) at least one disrupted alcohol dehydrogenase gene comprises a first peptide. In some embodiments the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163). In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a second peptide. In some embodiments the second peptide has the sequence QYATADAVQAA (SEQ ID NO: 158). In some embodiments the second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164). In some embodiments the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a third peptide. In some embodiments the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments, the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide. In some embodiments the fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of yeast species of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted and wherein at least one disrupted alcohol dehydrogenase gene comprises a first peptide. In some embodiments the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163). In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a second peptide. In some embodiments the second peptide has the sequence QYATADAVQAA (SEQ ID NO: 158). In some embodiments the second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164). In some embodiments the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165).

In some embodiments, the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a third peptide. In some embodiments the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide. In some embodiments the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments, the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide. In some embodiments said fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B, alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the β-oxidation pathway has also been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cyocrhrome P450s have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida in which endogenous cyocrhrome P450s have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a yeast strain, a species of Candida, or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of a fatty acid substrate (e.g., a α-carboxyl-ω-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an α,ω-dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof) have been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, or one or more disrupted alcohol oxidases, or one or more disrupted alcohol dehydrogenases present in the strain of yeast will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, one or more disrupted alcohol oxidases, and one or more disrupted alcohol dehydrogenases will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO1, FAO1B, FAO2 and FAO2B, alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the β-oxidation pathway has also been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cyocrhrome P450s have been disrupted.

In some embodiments, a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida in which endogenous cyocrhrome P450s have been disrupted.

To achieve novel phenotypes of Candida, it may be advantageous to modify the activity of a polypeptide by altering its sequence and to test the effect of the polypeptide with altered sequence within the yeast. A preferred method for testing the effect of sequence changes in a polypeptide within yeast is to introduce a plurality of genes of known sequence, each encoding a unique modified polypeptide, into the same genomic location in a plurality of strains.

The isocitrate lyase promoter from Candida tropicalis has been shown to be an inducible promoter in both Saccharomyces cerevisiae and E. coli (Atomi et al., 1995, Arch Microbiol: 163, 322-8; Umemura et al., 1995, Appl Microbiol Biotechnol: 43, 489-92.) When expressed in S. cerivisiae, the isocitrate lyase gene was found to be inducible by acetate, glycerol, lactate, ethanol, or oleate. Ethanol is interesting from the perspective that is a relatively cheap inducer and oleate for the fact that it is a potential substrate for the system for converting fatty acids to omega hydroxy fatty acids. Inducible expression of the Candida tropicalis ICL gene was found to be high in S. cerivisiae (as much as 30% of soluble protein), indicating that it may serve as a strong inducible promoter in C. tropicalis.

To insert genes under control of the isocitrate lyase promoter a genomic insertion construct of the form shown in FIG. 21 was synthesized. The sequence used for the sequence of promoter 1 was that of the Candida tropicalis isocitrate lyase promoter, given as SEQ ID NO: 62. This promoter has a BsiWI site that can be used to linearize the construct for subsequent insertion into the Candida tropicalis genome. The sequence used for transcription terminator 1 was that of the Candida tropicalis isocitrate lyase terminator, given as SEQ ID NO: 63. The sequence used for Promoter 2 was the TEF1 promoter, given as SEQ ID NO: 64. The sequence used for the bacterial promoter was the EM7 promoter, given as SEQ ID NO: 65. The sequence used for the selectable marker was the zeocin resistance gene, a version optimized for expression in Candida tropicalis is given as SEQ ID NO: 66. The sequence use for Transcription terminator 2 was the CYC1 transcription terminator, given as SEQ ID NO: 67. The sequence used as the bacterial origin of replication was the pUC origin, given as SEQ ID NO: 68. A genomic integration vector with these components is represented graphically as FIG. 23.

7.6.1. Insertion of CYP52A17 Under Control of the Isocitrate Lyase Promoter

A construct for expressing Candida tropicalis cytochrome P450 CYP52A17 under the control of the isocitrate lyase promoter was made by cloning the sequence of a gene encoding Candida tropicalis cytochrome P450 CYP52A17 (given as SEQ ID NO: 69) into a vector of the form shown in FIG. 23. The sequence of the complete vector is given as SEQ ID NO: 70.

The vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI. Candida tropicalis strains were transformed with the construct as described in Section 7.1.2, except that 100 μg/ml of zeocin was used instead of 200 μg/ml nourseothricin as the selective antibiotic. Genomic DNA was prepared and tested for the presence of the integrated DNA as described in Section 7.1.3.

Candida tropicalis strain DP201 was prepared by integration of the construct shown as SEQ ID NO: 70 into the genome of strain DP186 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. DP428 was prepared by integration of the construct shown as SEQ ID NO: 70 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. Sequences of oligonucleotide primers for analysis of strains were:

ICL-IN-F1: (SEQ ID NO: 124) GGATCCGTCTGAAGAAATCAAGAACC 1758R2: (SEQ ID NO: 125) TGGTGTAGGCCAATAATTGCTTAATGATATACAAAACTGGCACCACAA 1758F2: (SEQ ID NO: 126) GAGCAATTGTTGGAATATTGGTACGTTGTGGTGCCAGTTTTGTATATCA 1758R34: (SEQ ID NO: 127) CGAACTTAACAATAGCACCGTCTTGCAAACACATGGTCAAGTTAGTTAA.

For strains DP201 and DP428 (integrants of SEQ ID NO: 70), PCR with primers ICL-IN-F1 and 1758R2 produces a 1609 base pair amplicon indicating that the construct has been integrated in the ICL promoter region; PCR with primers 1758F2 and 1758R34 produces a 1543 base pair amplicon indicating that CYP52A17 has been integrated. Neither primer pair produces an amplicon from the parental strains DP186 or DP421.

7.6.2. Insertion of CYP52A13 Under Control of the Isocitrate Lyase Promoter

A construct for expressing Candida tropicalis cytochrome P450 CYP52A13 under the control of the isocitrate lyase promoter was made by cloning the sequence of a gene encoding Candida tropicalis cytochrome P450 CYP52A13 (given as SEQ ID NO: 71) into a vector of the form shown in FIG. 23. The sequence of the complete vector is given as SEQ ID NO: 72.

The vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI. Candida tropicalis strains were transformed with the construct as described in Section 7.1.2, except that 100 μg/ml of zeocin was used instead of 200 μg/ml nourseothricin as the selective antibiotic. Genomic DNA was prepared and tested for the presence of the integrated DNA as described in Section 7.1.3.

Candida tropicalis strain DP522 was prepared by integration of the construct shown as SEQ ID NO: 72 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. Sequences of oligonucleotide primers for analysis of strains were:

ICL-IN-F1: (SEQ ID NO: 124) 4082R2: (SEQ ID NO: 128) CGATTAAGGCCAATGGAACAATGACGTACCACTTAGTAAAGTAGGTA 4082F2: (SEQ ID NO: 129) CATGACTGTTCACGACATTATTGCTACCTACTTTACTAAGTGGTACGTC 4082R34: (SEQ ID NO: 130) ACATTTCAATATTAGCACCGTCAAATAATGACATGGTCAAATGGGACA

For strain DP522 (integration of SEQ ID NO: 72), PCR with primers ICL-IN-F1 and 4082R2 produces a 1600 base pair amplicon indicating that the construct has been integrated in the ICL promoter region; PCR with primers 4082F2 and 4082R34 produces a 1565 base pair amplicon indicating that CYP52A13 has been integrated. Neither primer pair produces an amplicon from the parental strain DP421.

7.6.3. Insertion of CYP52A12 Under Control of the Isocitrate Lyase Promoter

A construct for expressing Candida tropicalis cytochrome P450 CYP52A12 under the control of the isocitrate lyase promoter was made by cloning the sequence of a gene encoding Candida tropicalis cytochrome P450 CYP52A12 (given as SEQ ID NO: 73) into a vector of the form shown in FIG. 23. The sequence of the complete vector is given as SEQ ID NO: 74.

The vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI. Candida tropicalis strains were transformed with the construct as described in Section 7.1.2, except that 100 μg/ml of zeocin was used instead of 200 μg/ml nourseothricin as the selective antibiotic. Genomic DNA was prepared and tested for the presence of the integrated DNA as described in Section 7.1.3.

Candida tropicalis strain DP526 was prepared by integration of the construct shown as SEQ ID NO: 74 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. Sequences of oligonucleotide primers for analysis of strains were:

ICL-IN-F1: (SEQ ID NO: 124) CYP52A12-R2: (SEQ ID NO: 131) ATCAATAATTTCCTGGGTTGCCAT CYP52A12-F1: (SEQ ID NO: 132) ATGGCAACCCAGGAAATTATTGAT CYP52A12-R1: (SEQ ID NO: 133) CTACATCTTGACAAAAACACCATCATT

For strain DP526 (integration of SEQ ID NO: 74), PCR with primers ICL-IN-F1 and 4082R2 produces a 1554 base pair amplicon indicating that the construct has been integrated in the ICL promoter region; PCR with primers 4082F2 and 4082R34 produces a 1572 base pair amplicon indicating that CYP52A12 has been integrated. Neither primer pair produces an amplicon from the parental strain DP421.

7.7. Deletion of POX Genes from Candida Tropicalis

Picataggio et al., 1991, Mol Cell Biol: 11, 4333-9, describe a system for the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase, which catalyze the first reaction in the β-oxidation pathway of fatty acids. An alternative method is to use the SAT-1 flipper.

7.7.1. Deletion of POX4 Alleles

The sequence of a gene encoding an acyl-coenzyme A oxidase II (PXP-4) of Candida tropicalis, POX4, is given as SEQ ID NO: 136. This sequence was used to design two “pre-targeting” constructs. The first pre-targeting construct is comprised of two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences are separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 bp stuffer fragment and an XhoI restriction site. The targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the first POX4 pre-targeting construct is given as SEQ ID NO: 137. Not shown in SEQ ID NO: 137 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli. The first pre-targeting sequence can be synthesized using standard DNA synthesis techniques well known in the art.

The second pre-targeting construct is comprised of two targeting sequences from the 5′ and 3′ end of the structural gene that lie internal to the 5′ and 3′ targeting sequences of the first pre-targeting construct. The targeting sequences are separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 by stuffer fragment and an XhoI restriction site. The targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the second POX4 pre-targeting construct is given as SEQ ID NO: 138. Not shown in SEQ ID NO: 138 but also present in the pre-targeting construct are a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli. The second pre-targeting sequence can synthesized using standard DNA synthesis techniques well known in the art.

Targeting sequences for deletion of the two POX4 alleles from the Candida tropicalis geneome can be prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating into the POX4 pre-targeting constructs (SEQ ID NO: 137 or SEQ ID NO: 138) from which the 20 bp stuffer has been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting first targeting construct for the deletion of the first allele of POX4 is given as SEQ ID NO: 139. The sequence of the resulting second targeting construct for the deletion of the second allele of POX4 is given is SEQ ID NO: 140. Because the POX4 targeting sequences of the second targeting construct lie internal to the targeting sequences of the first targeting construct, use of the first targeting construct to delete the first POX4 allele assures that use of the second targeting construct is specific for the second POX4 allele since the targeting sequences of the second targeting construct no longer exist in the first deleted allele.

Analysis of integrants and excisants can be performed as described in Section 7.1. Sequences of oligonucleotide primers for the analysis of strains are:

POX4-IN-L: (SEQ ID NO: 141) ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAG POX4-IN-R: (SEQ ID NO: 142) TTACTTGGACAAGATAGCAGCGGTTTC SAT1-R: (SEQ ID NO:79) TGGTACTGGTTCTCGGGAGCACAGG SAT1-F: (SEQ ID NO: 80) CGCTAGACAAATTCTTCCAAAAATTTTAGA

7.7.2. Deletion of POX5 Alleles

The sequence of a gene encoding an acyl-coenzyme A oxidase I (PXP-5) of Candida tropicalis, POX5, is given as SEQ ID NO: 143. This sequence was used to design two “pre-targeting” constructs. The first pre-targeting construct is comprised of two targeting sequences from the 5′ and 3′ end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 by stuffer fragment and an XhoI restriction site. The targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the first POX5 pre-targeting construct is given as SEQ ID NO: 144. Not shown in SEQ ID NO: 144 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli. The first pre-targeting sequence can be synthesized using standard DNA synthesis techniques well known in the art.

The second pre-targeting construct is comprised of two targeting sequences from the 5′ and 3′ end of the structural gene that lie internal to the 5′ and 3′ targeting sequences of the first pre-targeting construct. The 5′ targeting sequence of the second pre-targeting construct is modified at position 248 (C248T) and 294 (G294A) to remove unwanted XhoI and BsmBI sites, respectively. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a NotI restriction site, a 20 by stuffer fragment and an XhoI restriction site. The targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the second POX5 pre-targeting construct is given as SEQ ID NO: 145. Not shown in SEQ ID NO: 145 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli. The second pre-targeting sequence can be synthesized using standard DNA synthesis techniques well known in the art.

Targeting sequences for deletion of the two POX5 alleles from the Candida tropicalis geneome were prepared by digesting the SAT-1 flipper (SEQ ID NO: 1) with restriction enzymes NotI and XhoI, and ligating into both of the POX5 pre-targeting constructs (SEQ ID NO 144 or 145) from which the 20 bp stuffer had been removed by digestion with restriction enzymes NotI and XhoI. The sequence of the resulting first targeting construct for the deletion of the first allele of POX5 is given as SEQ ID NO: 146. The sequence of the resulting second targeting construct for the deletion of the second allele of POX5 is given is SEQ ID NO: 147. Because the POX5 targeting sequences of the second targeting construct lie internal to the targeting sequences of the first targeting construct, use of the first targeting construct to delete the first POX5 allele assures that use of the second targeting construct is specific for the second POX5 allele since the targeting sequences of the second targeting construct no longer exist in the first deleted allele.

Analysis of integrants and excisants can be performed as described in section 7.1. Sequences of oligonucleotide primers for the analysis of strains are:

POX5-IN-L: (SEQ ID NO: 148) ATGCCTACCGAACTTCAAAAAGAAAGAGAA POX5-IN-R: (SEQ ID NO: 149) TTAACTGGACAAGATTTCAGCAGCTTCTTC SAT1-R: (SEQ ID NO: 79) TGGTACTGGTTCTCGGGAGCACAGG SAT1-F: (SEQ ID NO: 80) CGCTAGACAAATTCTTCCAAAAATTTTAGA

7.8. Insertion of Genes into the Genome of Candida

To achieve novel phenotypes in yeasts of the genus Candida (e.g., Candida tropicalis), including biotransformations of compounds by Candida tropicalis, including chemical conversions not previously obtained, or increased rates of conversion of one or more substrates to one or more products, or increased specificity of conversion of one or more substrates to one or more products, or increased tolerance of a compound by the yeast, or increased uptake of a compound by the yeast, it may be advantageous to incorporate a gene encoding a polypeptide into the genome of the yeast. Expression of the polypeptide in the yeast then allows the phenotype of the yeast to be modified.

To achieve novel phenotypes of Candida, it may be advantageous to modify the activity of a polypeptide by altering its sequence and to test the effect of the polypeptide with altered sequence within the yeast. A preferred method for testing the effect of sequence changes in a polypeptide within yeast is to introduce a plurality of genes of known sequence, each encoding a unique modified polypeptide, into the same genomic location in a plurality of strains.

The isocitrate lyase promoter from Candida tropicalis has been shown to be an inducible promoter in both Saccharomyces cerevisiae and E. coli as described in Atomi H. et al, 1995 Arch Microbiol. 163:322-8; Umemura K. et al, 1995 Appl Microbiol Biotechnol. 43:489-92; Kanai T. et al, 1996 Appl Microbiol Biotechnol. 44:759-65. The paper by Atomi H. et al, 1995 Arch Microbiol. 163:322-8, identified the sequence between bases −394 and −379 of the promoter as a promoter that regulates the isocitrate lyase promoter in the yeast Saccharomyces cerevisiae. The DNA sequence of an isocitrate lyase promoter from Candida tropicalis from base −394 to −1 is given as SEQ ID NO 161. Inducible expression of the Candida tropicalis ICL gene was found to be high in S. cerivisiae (as much as 30% of soluble protein), indicating that it may serve as a strong inducible promoter in C. tropicalis. The sequence of an isocitrate lyase promoter that has been used to drive expression of a protein in the yeast Saccharomyces cerevisiae is given as SEQ ID NO: 171. To insert genes under control of the isocitrate lyase promoter a genomic insertion construct of the form shown in FIG. 21 was synthesized. A genomic integration vector with these components is represented graphically as FIG. 23.

In some embodiments, a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises an isocitrate lyase promoter, in some embodiments a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises the sequence shown as SEQ ID NO: 62, in some embodiments a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises the sequence shown as SEQ ID 161, in some embodiments a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises a sequence that is 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence shown as SEQ ID 161. In some embodiments a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises a sequence of sufficient length and identity to the isocitrate lyase promoter to ensure integration at that locus; in some embodiments said construct comprises at least 100 contiguous base pairs or at least 200 contiguous base pairs or at least 300 contiguous base pairs or at least 400 contiguous base pairs or at least 500 contiguous base pairs of the sequence shown as SEQ ID NO: 62 or to the sequence shown as SEQ ID NO: 171; in some embodiments the construct comprises at least 100 contiguous base pairs or at least 200 contiguous base pairs or at least 300 contiguous base pairs or at least 400 contiguous base pairs or at least 500 contiguous base pairs that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence shown as SEQ ID NO: 62 or to the sequence shown as SEQ ID NO: 171.

Genes may also be inserted into the genome of yeasts of the genus Candida under control of other promoters by constructing analogous constructs to the one shown schematically in FIG. 21. Of particular utility may be the promoters for alcohol dehydrogenase genes, which are known to be highly expressed in other yeasts such as Saccharomyces cerevisiae. A construct for integrating into an alcohol dehydrogenase gene locus could also have an advantage in embodiments in which it is desirable to disrupt the alcohol dehydrogenase gene itself. In these cases it would be unnecessary to know the full sequence of the promoter: replacing all or a part of the coding sequence of the gene to be disrupted with the coding sequence of the gene to be inserted would be sufficient.

In some embodiments a construct for integration of a gene into the Candida genome with the aim of expressing a protein from that gene comprises a promoter from an alcohol dehydrogenase gene or a promoter from a cytochrome P450 gene, or a promoter for a fatty alcohol oxidase gene.

In some embodiments of the invention a gene encoding a polypeptideis integrated under control of an isocitrate lyase promoter, an alcohol dehydrogenase promoter, a fatty alcohol oxidase promoter or a cytochrome P450 promoter into a strain of Candida tropicalis in which one or more of the alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B1B and ADH-B11 have been disrupted. In some embodiments of the invention a gene encoding a polypeptideis integrated under control of an isocitrate lyase promoter, an alcohol dehydrogenase promoter, a fatty alcohol oxidase promoter or a cytochrome P450 promoter into a yeast strain of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides within the coding region, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence or at least 75% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 70% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 65% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 60% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence with one of the Candida tropicalis genes ADH-A4 (SEQ ID NO: 39), ADH-B4 (SEQ ID NO: 42), ADH-A10 (SEQ ID NO: 40), ADH-A10B (SEQ ID NO: 56), ADH-B11 (SEQ ID NO: 43). In some embodiments of the invention a gene encoding a polypeptideis integrated under control of an isocitrate lyase promoter, an alcohol dehydrogenase promoter, a fatty alcohol oxidase promoter or a cytochrome P450 promoter into a yeast strain of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase comprises a first peptide. In some embodiments the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments, the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a second peptide. In some embodiments the second peptide has the sequence QYATADAVQAA (SEQ ID NO: 158). In some embodiments the second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164). In some embodiments the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165).

In some embodiments the disrupted alcohol dehydrogenase sequence, predicted from translation of the gene that encodes it, comprises a third peptide. In some embodiments the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide. In some embodiments the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments, the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMG (SEQ ID NO: 169). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).

In some embodiments the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide. In some embodiments the fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).

Insertion of the gene encoding mCherry under control of the isocitrate lyase promoter

A construct for expressing mCherry (Shaner N C, Campbell R E, Steinbach P A, Giepmans B N, Palmer A E, Tsien R Y. (2004) Nat Biotechnol. 22:1567-72) under the control of the C. tropicalis isocitrate lyase promoter (given as SEQ ID NO: 62) was made by cloning the sequence of a gene encoding mCherry (given as SEQ ID NO: 75) into a vector of the form shown in FIG. 23 with the mCherry open reading frame in the position indicated by the element labeled “Gene for expression”. The sequence of the complete vector is given as SEQ ID NO: 76.

The vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI. Candida tropicalis strain DP186 (Table 3) was transformed with the construct or a no DNA control as described in Section 7.1.2, except that 200, 400 or 600 μg/ml of zeocin were used instead of 200 μg/ml nourseothricin as the selective antibiotic. Following ˜48 hours at 30° C. and an additional 24 hours at room temperature, 10 large red colonies were observed amongst a virtually confluent background of small white colonies on YPD agar plates with 200 ug/ml zeocin. Likewise, following 48 hours at 30° C. and an additional 48 hours at room temperature, large red colonies were observed on the 400 and 600 ug/ml zeocin YPD agar plates amongst a background of smaller white colonies. No red colonies were observed on plates transformed with the no DNA control. A total of 8 large, red colonies were isolated and selected for further characterization (see FIG. 27). Genomic DNA was prepared from the isolates and tested for the presence of the integrated mCherry DNA at the isocitriate lyase promter as described in Section 7.1.3. All 8 tested positive for mCherry integration at the isocitrate lyase promoter demonstrating that expression of genes other than isocitrate lyase can be driven in C. tropicalis using this promoter

One of the eight isolates, Candida tropicalis strain DP197 (Table 3), was prepared by integration of the construct shown as SEQ ID NO: 75 into the genome of strain DP186 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase.

ICL-IN-F1: (SEQ ID NO: 124) GGATCCGTCTGAAGAAATCAAGAACC 1759R33: (SEQ ID NO: 150) ACCTTAAAACGCATAAATTCCTTGATGATTGCCATGTTGTCTTCTTCA

For strain DP197 (integrant of SEQ ID NO: 75), PCR with primers ICL-IN-F1 and 1759R33 produces a 1592 base pair amplicon indicating that the construct has been integrated in the ICL promoter region. The primer pair does not produces an amplicon from the parental strain DP186.

8. CONVERSION OF FATTY ACIDS USING MODIFIED STRAINS OF CANDIDA TROPICALIS 8.1. Analytical Methods 8.1.1. GC-MS for Identification of Fatty Acids, Omega-Hydroxy Fatty Acids and Diacids

Gas chromatography/mass spectrometry (GC/MS) analysis was performed at 70 eV with ThermoFinnigan TraceGC Ultra gas chromatograph coupled with Trace DSQ mass spectrometer. Products were esterified with BF₃ in methanol (10%, w/w) at 70° C. for 20 min, and further silylation of the methyl esters with HMDS/TMCS/Pyridine at 70° C. for 10 min when needed. The experiments were carried out with injector, ion source and interface temperature of 200° C., 250° C. and 280° C., respectively. Samples in hexane (1 μl) were injected in PTV split mode and run on a capillary column (Varian CP8944 VF-SMS, 0.25 mm×0.25 um×30 m). The oven temperature was programmed at 120° C. for one minute increasing to 260° C. at the rate of 20° C./minute, and then to 280° C. at the rate of 4.0° C./minute.

8.1.2. LC-MS for Measurement of Fatty Acids, Omega-Hydroxy Fatty Acids and Diacids

The concentration of omega-hydroxy fatty acids and diacids during biotransformation was measured by liquid chromatography/mass spectrometry (LC/MS) with purified products as standards. The solvent delivery system was a Waters Alliance 2795 Separation Module (Milford, Mass., USA) coupled with a Waters 2996 photodiode array detector and Waters ZQ detector with an electron spray ionization mode. The separation was carried on a reversed-phase column with a dimension of 150×4.6 mm and particle size of 5 nm. The mobile phase used for separation contained 10% H₂O, 5% acetonitrile, 5% Formic acid solution (1% in water) and 80% methanol.

8.1.3. NMR for Characterization of Omega-Hydroxyfatty Acids and Diacids

Proton (¹H) and ¹³C-NMR spectra were recorded on a Bruker DPX300 NMR spectrometer at 300 MHz. The chemical shifts (ppm) for ¹H-NMR were referenced relative to tetramethylsilane (TMS, 0.00 ppm) as the internal reference.

8.2. Oxidation of Fatty Acids by Candida Tropicalis Strains Lacking Four CYP52A P450S

We compared the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17 and CYP52A18 (DP174) constructed in Section 7.2 with the starting strain (DP1) for their abilities to oxidize fatty acids. To engineer P450s for optimal oxidation of fatty acids or other substrates it is advantageous to eliminate the endogenous P450s whose activities may mask the activities of the enzymes being engineered. We tested Candida tropicalis strains DP1 and DP174 (genotypes given in Table 3) to determine whether the deletion of the four CYP52 P450S had affected the ability of the yeast to oxidize fatty acids.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 30 g/l glucose. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 60 g/l glucose in a 125 ml flask, and grown at 30° C. and 250 rpm for 12 hours. Substrates were added and shaking was continued at 30° C. and 250 rpm. We then tested the conversion of C14 fatty acid substrates as shown in FIG. 13. FIG. 13 parts A and B show that the starting strain DP1 converts methyl myristate to ω-hydroxy myristate and to the C14 diacid produced by oxidation of the ω-hydroxy myristate over a 48 hour time course, while the quadruple P450 deletion strain DP174 can effect almost no detectable conversion. FIG. 13 parts C and D show that the starting strain DP1 converts methyl myristate and sodium myristate to ω-hydroxy myristate and to the C14 diacid produced by oxidation of the ω-hydroxy myristate after 48 hours, while the quadruple P450 deletion strain DP174 effects almost no detectable conversion of these substrates.

These results confirm that at least one of the four Candida tropicalis cytochrome

P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18 is required for hydroxylation of fatty acids, consistent with the schematic representation of Candida tropicalis fatty acid metabolism pathways shown in FIG. 12. Further it shows that strain DP174 is an appropriate strain to use for testing of engineered cytochrome P450s, since it has essentially no ability to oxidize fatty acids without an added P450.

8.3. Oxidation of Ω-Hydroxy Fatty Acids by Candida Tropicalis Strains Lacking Four CYP52A P450S

We compared the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17 and CYP52A18 (DP174) constructed in Section 7.2 with the starting strain (DP1) for their abilities to oxidize ω-hydroxy fatty acids. To engineer a strain for the production of ω-hydroxy fatty acids it is desirable to eliminate enzymes from the cell that can oxidize ω-hydroxy fatty acids. It is possible to determine whether other enzymes involved in oxidation of ω-hydroxy fatty acids are present in the strain by feeding it ω-hydroxy fatty acids in the media. If there are enzymes present that can oxidize ω-hydroxy fatty acids, then the strain will convert ω-hydroxy fatty acids fed in the media to α,ω-dicarboxylic acids.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g/l glycerol in a 125 ml flask, and grown at 30° C. and 280 rpm for 12 hours. We then tested the conversion of C12 and C16 ω-hydroxy fatty acid substrates by adding these substrates to independent flasks at final concentrations of 5 g/l and the pH was adjusted to between 7.5 and 8 and shaking was continued at 30° C. and 250 rpm. Samples were taken at the times indicated, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy fatty acids and α,ω-diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). The results are shown in Table 5.

TABLE 5 Oxidation of ω-hydroxy fatty acids by Candida tropicalis Ω-HYDROXY FATTY ACID DIACID DIACID SUBSTRATE REACTION PRODUCED BY PRODUCED BY CHAIN LENGTH TIME DP1 (G/L) DP174 (G/L) C12 60 hours 5.6 5.2 C16 60 hours 1.4 0.8 C12 24 hours 5.4 5 C12 48 hours 6 6.7 C12 72 hours 6.2 6.5 C16 24 hours 2.3 0.9 C16 48 hours 2.4 1.7 C16 72 hours 2.8 1.8

These results show that at least one enzyme capable of oxidizing ω-hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18.

8.4. Oxidation of Ω-Hydroxy Fatty Acids by Candida Tropicalis Strains Lacking Four CYP52A P450S and Four Fatty Alcohol Oxidases

We compared the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18 and FAO1 (DP186) constructed in Section 7.3 with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A and FAO2B (DP258 and DP259) for their abilities to oxidize ω-hydroxy fatty acids. To engineer a strain for the production of ω-hydroxy fatty acids it is desirable to eliminate enzymes from the cell that can oxidize ω-hydroxy fatty acids. It is possible to determine whether other enzymes involved in oxidation of ω-hydroxy fatty acids are present in the strain by feeding it ω-hydroxy fatty acids in the media. If there are enzymes present that can oxidize ω-hydroxy fatty acids, then the strain will convert ω-hydroxy fatty acids fed in the media to α,ω-dicarboxylic acids.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/l, KH₂PO₄ 9.3 g/1) plus 20 g/l glycerol. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g/l glycerol in a 125 ml flask, and grown at 30° C. and 250 rpm for 12 hours. We then tested the conversion of C12 and C16 ω-hydroxy fatty acid substrates by adding these substrates to independent flasks at final concentrations of 5 g/l and the pH was adjusted to between 7.5 and 8 and shaking was continued at 30° C. and 250 rpm. Samples were taken after 24 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy fatty acids and α,ω-diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). As shown in FIG. 15 most of the hydroxy fatty acids are converted to diacid after 24 hours. These results show that at least one enzyme capable of oxidizing ω-hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A and FAO2B.

8.5. Oxidation of Ω-Hydroxy Fatty Acids by Candida Tropicalis Strains Lacking Six CYP52A P450S and Four Fatty Alcohol Oxidases

We compared the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18 and FAO1 (DP186) constructed in Section 7.2 with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12 and CYP52A12B (DP283 and DP284) for their abilities to oxidize ω-hydroxy fatty acids. To engineer a strain for the production of ω-hydroxy fatty acids it is desirable to eliminate enzymes from the cell that can oxidize ω-hydroxy fatty acids. It is possible to determine whether other enzymes involved in oxidation of ω-hydroxy fatty acids are present in the strain by feeding it ω-hydroxy fatty acids in the media. If there are enzymes present that can oxidize ω-hydroxy fatty acids, then the strain will convert ω-hydroxy fatty acids fed in the media to α,ω-dicarboxylic acids.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 16 hours 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g/l glycerol in a 125 ml flask, and grown at 30° C. and 250 rpm for 12 hours. We then tested the conversion of C12 and C16 ω-hydroxy fatty acid substrates by adding these substrates to independent flasks at final concentrations of 5 g/l and the pH was adjusted to between 7.5 and 8 and shaking was continued at 30° C. and 250 rpm. Samples were taken after 24 hours, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy fatty acids and α,ω-diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). As shown in FIG. 16 most of the C12 hydroxy fatty acids and a substantial fraction of the C16 hydroxy fatty acids are converted to diacid after 24 hours. These results show that at least one enzyme capable of oxidizing ω-hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17, CYP52A18, CYP52A12, CYP52A12B, FAO1, FAO1B, FAO2A and FAO2B.

8.6. Oxidation of Ω-Hydroxy Fatty Acids by Candida Tropicalis Strains Lacking Six CYP52A P450S, Four Fatty Alcohol Oxidases and Five Alcohol Dehydrogenases

We compared the Candida tropicalis strain DP1 with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12 and CYP52A12B (DP283) and the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B and ADH-A10 (DP415) for their abilities to oxidize ω-hydroxy fatty acids. To engineer a strain for the production of ω-hydroxy fatty acids it is desirable to eliminate enzymes from the cell that can oxidize ω-hydroxy fatty acids. It is possible to determine whether other enzymes involved in oxidation of ω-hydroxy fatty acids are present in the strain by feeding it ω-hydroxy fatty acids in the media. If there are enzymes present that can oxidize ω-hydroxy fatty acids, then the strain will convert ω-hydroxy fatty acids fed in the media to α,ω-dicarboxylic acids.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 18 hours the preculture was diluted in fresh media to A₆₀₀=1.0. This culture was shaken until the A₆₀₀ reached between 5.0 and 6.0. Biocatalytic conversion was initiated by adding 5 ml culture to a 125 ml flask together with 50 mg of ω-hydroxy lauric acid, and pH adjusted to ˜7.5 with 2M NaOH. Samples were taken at the times indicated, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). As shown in FIG. 19 Part A, the cell growth was almost identical for the 3 strains. Strain DP415 produced much less α,ω-dicarboxy laurate than the other two strains, however, as shown in FIG. 19 part B.

These results show that a significant reduction in the ability of Candida tropicalis to oxidize ω-hydroxy fatty acids can be reduced by deleting genes encoding CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B and ADH-A10.

8.7. Oxidation of Ω-Hydroxy Fatty Acids by Candida Tropicalis Strains Lacking Six CYP52A P450S, Four Fatty Alcohol Oxidases and Eight Alcohol Dehydrogenases

We compared the Candida tropicalis strain DP1 with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4 and ADH-A4B (DP390), the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B and ADH-A10 (DP415), the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 (DP417 and DP421), the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B and ADH-B11 (DP423), the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11 and ADH-B11B (DP434 and DP436) for their abilities to oxidize ω-hydroxy fatty acids. To engineer a strain for the production of ω-hydroxy fatty acids it is desirable to eliminate enzymes from the cell that can oxidize ω-hydroxy fatty acids. It is possible to determine whether other enzymes involved in oxidation of ω-hydroxy fatty acids are present in the strain by feeding it ω-hydroxy fatty acids in the media. If there are enzymes present that can oxidize ω-hydroxy fatty acids, then the strain will convert ω-hydroxy fatty acids fed in the media to α,β-dicarboxylic acids.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glycerol. After 18 hours the preculture was diluted in fresh media to A₆₀₀=1.0. This culture was shaken until the A₆₀₀ reached between 5.0 and 6.0. Biocatalytic conversion was initiated by adding 5 ml culture to a 125 ml flask together with 50 mg of ω-hydroxy lauric acid, and pH adjusted to ˜7.5 with 2M NaOH. Samples were taken at the times indicated, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of α,ω-diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). As shown in FIG. 20, a significant reduction in the ability of Candida tropicalis to oxidize ω-hydroxy fatty acids can be obtained by deleting genes encoding alcohol dehydrogenases in strains lacking some cytochrome P450s and fatty alcohol oxidases.

8.8. Oxidation of Methyl Myristate by Candida Tropicalis Strains Lacking Six CYP52A P450S, Four Fatty Alcohol Oxidases and Six Alcohol Dehydrogenases with a Single CYP52A P450 Added Back Under Control of the ICL Promoter

We compared the Candida tropicalis strain DP1 with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18 and FAO1 and with CYP52A17 added back under control of the isocitrate lyase promoter (DP201) and with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 and with CYP52A17 added back under control of the isocitrate lyase promoter (DP428) for their abilities to oxidize methyl myristate.

Cultures of the yeast strains were grown at 30° C. and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/1, KH₂PO₄ 9.3 g/l) plus 20 g/l glucose plus 5 g/l ethanol. After 18 hours 3 ml of preculture was added to 27 ml fresh media F plus 20 g/l glucose plus 5 g/l ethanol in a 500 ml flask, and grown at 30° C. and 250 rpm for 20 hours before addition of substrate. Biocatalytic conversion was initiated by adding 40 g/l of methyl myristate, the pH was adjusted to ˜7.8 with 2M NaOH. The culture was pH controlled by adding 2 mol/l NaOH every 12 hours, glycerol was fed as cosubstrate by adding 500 g/l glycerol and ethanol was fed as a inducer by adding 50% ethanol every 12 hours. Samples were taken at the times indicated, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy myristate and α,ω-dicarboxymyristate were measured by LC-MS (liquid chromatography mass spectroscopy).

As shown in FIG. 24, strains DP1 and DP201 both produce significant levels of tetradecanedioic acid (the α,ω-diacid) and negligible levels of ω-hydroxy myristic acid. In contrast, under these conditions strain DP428 produces approximately five-fold less tetradecanedioic acid, while converting nearly 70% of the methyl myristate to ω-hydroxy myristic acid after 60 hours. This shows that elimination of one or more of the genes FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 prevents the over-oxidation of the fatty acid myristic acid by Candida tropicalis, and that the presence of CYP52A17 under control of the isocitrate lyase promoter in this strain background produces a strain that can convert methyl myristate to ω-hydroxy myristic acid, but that does not over-oxidize the product to tetradecanedioic acid.

8.9. Oxidation of Methyl Myristate by an Engineered Candida tropicalis Strain in a Fermentor

We compared the production of ω-hydroxy myristic acid and α,ω-tetradecanoic acid by a Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 and with CYP52A17 added back under control of the isocitrate lyase promoter (DP428).

C. tropicalis DP428 was taken from a glycerol stock or fresh agar plate and inoculated into 500 ml shake flask containing 30 mL of YPD medium (20 g/l glucose, 20 g/l peptone and 10 g/l yeast extract) and shaken at 30° C., 250 rpm for 20 h. Cells were collected by centrifugation and re-suspended in FM3 medium for inoculation. (FM3 medium is 30 g/l glucose, 7 g/l ammonium sulfate, 5.1 g/l potassium phosphate, monobasic, 0.5 g/l magnesium sulfate, 0.1 g/l calcium chloride, 0.06 g/l citric acid, 0.023 g/l ferric chloride, 0.0002 g/l biotin and 1 ml/l of a trace elements solution. The trace elements solution contains 0.9 g/l boric acid, 0.07 g/l cupric sulfate, 0.18 g/l potassium iodide, 0.36 g/l ferric chloride, 0.72 g/l manganese sulfate, 0.36 g/l sodium molybdate, 0.72 g/l zinc sulfate.) Conversion was performed by inoculating 15 ml of preculture into 135 ml FM3 medium, methyl myristate was added to 20 g/l and the temperature was kept at 30° C. The pH was maintained at 6.0 by automatic addition of 6 M NaOH or 2 M H₂SO₄ solution. Dissolved oxygen was kept at 70% by agitation and O₂-cascade control mode. After 6 hours growth, ethanol was fed into the cell culture to 5 g/l. During the conversion phase, 80% glycerol was fed as ω-substrate by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%). Every 12 hours, ethanol was added into cell culture to 2 g/l, and methyl myristate was added to 40 g/l until the total methyl myristate added was 140 g/l (i.e. the initial 20 g/l plus 3 subsequent 40 g/l additions). Formation of products was measured at the indicated intervals by taking samples and acidifying to pH ˜1.0 by addition of 6 N HCl; products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy myristate and α,ω-dicarboxymyristate were measured by LC-MS (liquid chromatography mass spectroscopy), as shown in FIG. 26. Under these conditions the strain produced a final concentration of 91.5 g/l ω-hydroxy myristic acid, with a productivity of 1.63 g/l/hr and a w/w ratio of ω-hydroxy myristic acid:tetradecanedioic acid of 20.3:1. This shows that elimination of one or more of the genes FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11 prevents the over-oxidation of the fatty acid myristic acid by Candida tropicalis, and that the presence of CYP52A17 under control of the isocitrate lyase promoter in this strain background produces a strain that can convert methyl myristate to ω-hydroxy myristic acid, but that does not over-oxidize the product to tetradecanedioic acid.

8.10. Oxidation of Methyl Myristate, Oleic Acid and Linoleic Acid by Engineered Candida Tropicalis Strains

We compared the fatty acid oxidizing activities of two Candida tropicalis strains which lack CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAO1, FAO1B, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10 and ADH-B11, one of which has CYP52A17 added back under control of the isocitrate lyase promoter (DP428) and one of which has CYP52A13 added back under control of the isocitrate lyase promoter (DP522).

Cultures of the yeast strains were grown at 30° C. in a DASGIP parallel fermentor containing 200 ml of media F (media F is peptone 3 g/l, yeast extract 6 g/l, yeast nitrogen base 6.7 g/l, sodium acetate 3 g/l, K₂HPO₄ 7.2 g/l, KH₂PO₄ 9.3 g/1) plus 30 g/l glucose. The pH was maintained at 6.0 by automatic addition of 6 M NaOH or 2 M H₂SO₄ solution. Dissolved oxygen was kept at 70% by agitation and O₂-cascade control mode. After 6 hour growth, ethanol was fed into the cell culture to 5 g/l. After 12 h growth, biocatalytic conversion was initiated by adding methyl myristate acid to 60 g/l or oleic acid to 60 g/l or linoleic acid to 30 g/l. During the conversion phase, 80% glycerol was fed as ω-substrate for conversion of methyl myristate and 500 g/l glucose was fed as ω-substrate for conversion of oleic acid and linoleic acid by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%). Every 12 hour, ethanol was added into cell culture to 2 g/l. Samples were taken at various times, cell culture was acidified to pH ˜1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ω-hydroxy fatty acids and α,ω-diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). As shown in FIG. 25, strains DP428 and DP522 were both able to produce ω-hydroxy fatty acids from these substrates, as well as some α,ω-diacids. FIG. 25 also shows that the different P450s had different preferences for the fatty acid substrates, and different propensities to oxidize the ω-hydroxy group.

9. DEPOSIT OF MICROORGANISMS

A living cultures of strain DP421 has been deposited with American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, on May 4, 2009, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent procedure.

10. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All publications, patents, patent applications, and databases mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent, patent application or database was specifically and individually indicated to be incorporated herein by reference.

11. EXEMPLARY EMBODIMENTS

The following are nonlimiting exemplary embodiments in accordance with the disclosed application:

Embodiment 1

A substantially pure Candida host cell for the biotransformation of a substrate to a product, wherein the Candida host cell is characterized by a first genetic modification class that comprises one or more genetic modifications that collectively or individually disrupt an alcohol dehydrogenase gene in the substantially pure Candida host cell.

Embodiment 2

The substantially pure Candida host cell of embodiment 1, wherein the substantially pure Candida host cell is genetically modified Candida glabrata, Candida zeylenoides, Candida lipolytica, Candida guillermondii, Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bituminiphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida floricola, Candida floris, Candida flosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida marls, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, or Candida zeylanoides.

Embodiment 3

The substantially pure Candida host cell of embodiment 2, wherein the substantially pure Candida host cell is genetically modified Candida tropicalis.

Embodiment 4

The substantially pure Candida host cell of embodiment 3, wherein the substantially pure Candida host cell is selected from the group consisting of DP428, DP522 and DP 527.

Embodiment 5

The substantially pure Candida host cell of embodiment 1, wherein the substantially pure Candida host cell is genetically modified Candida tropicalis and wherein the alcohol dehydrogenase gene is selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11, and ADH-B11B.

Embodiment 6

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 7

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 8

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 9

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has at least 90 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 10

The substantially pure Candida host cell of embodiment 9, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 11

The substantially pure Candida host cell of embodiment 9, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 12

The substantially pure Candida host cell of embodiment 9, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 13

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has 100 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 14

The substantially pure Candida host cell of embodiment 13, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 15

The substantially pure Candida host cell of embodiment 13, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 16

The substantially pure Candida host cell of embodiment 13, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 17

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least one peptide selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 18

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least two peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 19

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least three peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 20

The substantially pure Candida host cell of embodiment 1, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least four peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 21

The substantially pure Candida host cell of embodiment 1, wherein the one or more genetic modifications in the first genetic modification class cause the alcohol dehydrogenase to have decreased function relative to the function of the wild-type counterpart, be nonfunctional, or have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.

Embodiment 22

The substantially pure Candida host cell of embodiment 1 that further comprises a second genetic modification class, wherein the second genetic modification class comprises an insertion of a first gene into the Candida host cell genome; wherein the first gene encodes

-   -   a protein that is not identical to a naturally occurring protein         in the substantially pure Candida host cell, or     -   a protein that is identical to a naturally occurring protein in         the substantially pure Candida host cell, but expression of the         gene is controlled by a promoter that is different from the         promoter that controls the expression of the naturally occurring         protein.

Embodiment 23

The substantially pure Candida host cell of embodiment 22, wherein the first gene encodes a desaturase, a lipase, a fatty alcohol oxidase, an alcohol dehydrogenase, a glycosyl transferase, a cytochrome P450, a cellulose, an exoglucanase, a cellobiohydrolase, an endoglucanase, a β-glucosidase, an α-amylase, a β-amylase, a γ-amylases, a glucoamylase, a maltogenase, a pullanase, an endo-β-xylanase, an α-glucuronidase, an α-arabinofuranosidase, a β-xylosidase, a β-mannanase, a β-mannosidase, a pectin lyase, an endo-polygalacturonase, an α-arabinofuranosidase, an α-galactosidase, a polymethylgalacturonase, a pectin depolymerase, a pectinase, an exopolygalacturanosidase hydrolase, an α-L-Rhamnosidase, an α-L-Arabinofuranosidase, a polymethylgalacturonate lyase, a polygalacturonate lyase, an exopolygalacturonate lyase, a peroxidase, a copper radical oxidase, an FAD-dependent oxidase, a multicopper oxidase, a lignin peroxidase or a manganese peroxidase that is

-   -   not identical to a naturally occurring protein in the         substantially pure Candida host cell; or     -   identical to a naturally occurring protein in the substantially         pure Candida host cell, but expression of the gene is controlled         by a promoter that is different from the promoter that controls         the expression of the naturally occurring protein.

Embodiment 24

The substantially pure Candida host cell of embodiment 22, wherein the first gene encodes a cytochrome P450 that is not identical to a naturally occurring cytochrome P450 in the substantially pure Candida host cell.

Embodiment 25

The substantially pure Candida host cell of embodiment 22, wherein the first gene is a gene listed in Table 4 other than a gene that naturally occurs in the substantially pure Candida host cell.

Embodiment 26

The substantially pure Candida host cell of embodiment 22, wherein the first gene has at least 40 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 27

The substantially pure Candida host cell of embodiment 22, wherein the first gene has at least 60 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 28

The substantially pure Candida host cell of embodiment 22, wherein the first gene has at least 80 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 29

The substantially pure Candida host cell of embodiment 22, wherein the first gene has at least 95 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 30

The substantially pure Candida host cell of embodiment 22, wherein the first gene is encoded by a nucleic acid that binds under conditions of high stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 31

The substantially pure Candida host cell of embodiment 22, wherein the first gene is encoded by a nucleic acid that binds under conditions of moderate stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 32

The substantially pure Candida host cell of embodiment 22, wherein the first gene is encoded by a nucleic acid that binds under conditions of low stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the substantially pure Candida host cell.

Embodiment 33

The substantially pure Candida host cell of embodiment 22, wherein the promoter is an isocitrate lyase promoter, a cytochrome P450 promoter, a fatty alcohol oxidase promoter or an alcohol dehydrogenase promoter in the Candida host cell genome.

Embodiment 34

The substantially pure Candida host cell of embodiment 33, wherein the promoter is an isocitrate lyase promoter.

Embodiment 35

The substantially pure Candida host cell of embodiment 1 that further comprises a third genetic modification class, wherein the third genetic modification class comprises one or more genetic modifications in the Candida host cell genome that collectively or individually disrupt

-   -   the β-oxidation pathway; or     -   a gene selected from the group consisting of a CYP52A type         cytochrome P450 and a fatty alcohol oxidase.

Embodiment 36

A method of using a genetically modified Candida cell for the biotransformation of a substrate to a product, wherein the genetically modified Candida cell is characterized by a first genetic modification class that comprises one or more genetic modifications that collectively or individually disrupt an alcohol dehydrogenase gene; and the method comprises fermenting the genetically modified Candida cell in a culture medium comprising a nitrogen source and a carbon source.

Embodiment 37

The method of embodiment 36, wherein the culture medium further comprises the substrate.

Embodiment 38

The method of embodiment 36, wherein the genetically modified Candida cell is genetically modified Candida glabrata, Candida zeylenoides, Candida lipolytica, Candida guillermondii, Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bituminiphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida floricola, Candida floris, Candida flosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida maris, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, or Candida zeylanoides.

Embodiment 39

The method of embodiment 36, wherein the genetically modified Candida cell is genetically modified Candida tropicalis.

Embodiment 40

The method of embodiment 36, wherein the genetically modified Candida cell is genetically modified Candida tropicalis and wherein the alcohol dehydrogenase is selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11, and ADH-B11B.

Embodiment 41

The method of embodiment 36, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 42

The method of embodiment 36, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 43

The method of embodiment 36, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 44

The method of embodiment 36, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has at least 90 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 45

The method of embodiment 44, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 46

The method of embodiment 44, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 47

The method of embodiment 44, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 48

The method of embodiment 36, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has 100 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 49

The method of embodiment 48, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 50

The method of embodiment 48, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 51

The method of embodiment 48, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 52

The method of embodiment 36, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least one peptide selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 53

The method of embodiment 36, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least two peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 54

The method of embodiment 36, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least three peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 55

The method of embodiment 36, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least four peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 56

The method of embodiment 36, wherein the one or more genetic modifications in the first genetic modification class cause the alcohol dehydrogenase to have decreased function relative to the function of the wild-type counterpart, be nonfunctional, or have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.

Embodiment 57

The method of embodiment 36, wherein the genetically modified Candida cell further comprises a second genetic modification class, wherein the second genetic modification class comprises an insertion of a first gene into the Candida host cell genome; wherein the first gene encodes

-   -   a protein that is not identical to a naturally occurring protein         in the Candida host cell, or     -   a protein that is identical to a naturally occurring protein in         the Candida host cell, but expression of the gene is controlled         by a promoter that is different from the promoter that controls         the expression of the naturally occurring protein.

Embodiment 58

The method of embodiment 57, wherein the first gene encodes a desaturase, a lipase, a fatty alcohol oxidase, an alcohol dehydrogenase, a glycosyl transferase, a cytochrome P450, a cellulose, an exoglucanase, a cellobiohydrolase, an endoglucanase, a β-glucosidase, an α-amylase, a β-amylase, a γ-amylases, a glucoamylase, a maltogenase, a pullanase, an endo-β-xylanase, an α-glucuronidase, an α-arabinofuranosidase, a β-xylosidase, a β-mannanase, a β-mannosidase, a pectin lyase, an endo-polygalacturonase, an α-arabinofuranosidase, an α-galactosidase, a polymethylgalacturonase, a pectin depolymerase, a pectinase, an exopolygalacturanosidase hydrolase, an α-L-Rhamnosidase, an α-L-Arabinofuranosidase, a polymethylgalacturonate lyase, a polygalacturonate lyase, an exopolygalacturonate lyase, a peroxidase, a copper radical oxidase, an FAD-dependent oxidase, a multicopper oxidase, a lignin peroxidase or a manganese peroxidase that is not identical to a naturally occurring protein in the Candida host cell or is identical to a naturally occurring protein in the Candida host cell, but expression of the gene is controlled by a promoter that is different from the promoter that controls the expression of the naturally occurring protein.

Embodiment 59

The method of embodiment 57, wherein the first gene encodes a cytochrome P450 that is not identical to a naturally occurring cytochrome P450 in the Candida host cell.

Embodiment 60

The method of embodiment 57, wherein the first gene is a gene listed in Table 4 other than a gene that naturally occurs in the Candida host cell.

Embodiment 61

The method of embodiment 57, wherein the first gene has at least 40 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 62

The method of embodiment 57, wherein the first gene has at least 60 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 63

The method of embodiment 57, wherein the first gene has at least 80 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 64

The method of embodiment 57, wherein the first gene has at least 95 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 65

The method of embodiment 57, wherein the first gene is encoded by a nucleic acid that binds under conditions of high stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 66

The method of embodiment 57, wherein the first gene is encoded by a nucleic acid that binds under conditions of moderate stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 67

The method of embodiment 57, wherein the first gene is encoded by a nucleic acid that binds under conditions of low stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 68

The method of embodiment 57, wherein the promoter is an isocitrate lyase promoter, a cytochrome P450 promoter, a fatty alcohol oxidase promoter or an alcohol dehydrogenase promoter in the Candida host cell genome.

Embodiment 69

The method of embodiment 57, wherein the promoter is an isocitrate lyase promoter.

Embodiment 70

The method of embodiment 36, wherein the genetically modified Candida cell further comprises a third genetic modification class, wherein the third genetic modification class comprises one or more genetic modifications in the Candida host cell genome that collectively or individually disrupt

-   -   the β-oxidation pathway; or     -   a gene selected from the group consisting of a CYP52A type         cytochrome P450 and a fatty alcohol oxidase.

Embodiment 71

A method for the biotransformation of a substrate to a product in a Candida host cell, the method comprising:

-   -   making one or more first genetic modifications in a first         genetic modification class to the Candida host cell, wherein the         first genetic modification class comprises one or more genetic         modifications that collectively or individually disrupt an         alcohol dehydrogenase gene; and     -   transforming the substrate to the product by fermenting the         Candida host cell in a culture medium comprising a nitrogen         source and a carbon source.

Embodiment 72

The method of embodiment 71, wherein the Candida host cell is genetically modified Candida glabrata, Candida zeylenoides, Candida lipolytica, Candida guillermondii, Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bituminiphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida floricola, Candida floris, Candida flosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida maxis, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, or Candida zeylanoides.

Embodiment 73

The method of embodiment 71, wherein the Candida host cell is genetically modified Candida tropicalis.

Embodiment 74

The method of embodiment 71, wherein the Candida host cell is selected from the group consisting of DP428, DP522 and DP 527.

Embodiment 75

The method of embodiment 71, wherein the Candida host cell is genetically modified Candida tropicalis and wherein the alcohol dehydrogenase gene is selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11, and ADH-B11B.

Embodiment 76

The method of embodiment 71, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 77

The method of embodiment 71, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 78

The method of embodiment 71, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 79

The method of embodiment 71, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has at least 90 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 80

The method of embodiment 79, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 81

The method of embodiment 79, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 82

The method of embodiment 79, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 83

The method of embodiment 71, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has 100 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 84

The method of embodiment 83, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 85

The method of embodiment 83, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 86

The method of embodiment 83, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 87

The method of embodiment 71, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least one peptide selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 88

The method of embodiment 71, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least two peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 89

The method of embodiment 71, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least three peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 90

The method of embodiment 71, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least four peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 91

The method of embodiment 71, wherein the one or more genetic modifications in the first genetic modification class cause the alcohol dehydrogenase to have decreased function relative to the function of the wild-type counterpart, be nonfunctional, or have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.

Embodiment 92

The method of embodiment 71 that further comprises a second genetic modification class, wherein the second genetic modification class comprises an insertion of a first gene into the Candida host cell genome; wherein the first gene encodes

-   -   a protein that is not identical to a naturally occurring protein         in the Candida host cell, or     -   a protein that is identical to a naturally occurring protein in         the Candida host cell, but expression of the gene is controlled         by a promoter that is different from the promoter that controls         the expression of the naturally occurring protein.

Embodiment 93

The method of embodiment 92, wherein the first gene encodes a desaturase, a lipase, a fatty alcohol oxidase, an alcohol dehydrogenase, a glycosyl transferase, a cytochrome P450, a cellulose, an exoglucanase, a cellobiohydrolase, an endoglucanase, a β-glucosidase, an α-amylase, a β-amylase, a γ-amylases, a glucoamylase, a maltogenase, a pullanase, an endo-β-xylanase, an α-glucuronidase, an α-arabinofuranosidase, a β-xylosidase, a β-mannanase, a β-mannosidase, a pectin lyase, an endo-polygalacturonase, an α-arabinofuranosidase, an α-galactosidase, a polymethylgalacturonase, a pectin depolymerase, a pectinase, an exopolygalacturanosidase hydrolase, an α-L-Rhamnosidase, an α-L-Arabinofuranosidase, a polymethylgalacturonate lyase, a polygalacturonate lyase, an exopolygalacturonate lyase, a peroxidase, a copper radical oxidase, an FAD-dependent oxidase, a multicopper oxidase, a lignin peroxidase or a manganese peroxidase that is

-   -   not identical to a naturally occurring protein in the Candida         host cell; or     -   identical to a naturally occurring protein in the Candida host         cell, but expression of the gene is controlled by a promoter         that is different from the promoter that controls the expression         of the naturally occurring protein.

Embodiment 94

The method of embodiment 92, wherein the first gene encodes a cytochrome P450 that is not identical to a naturally occurring cytochrome P450 in the Candida host cell.

Embodiment 95

The method of embodiment 92, wherein the first gene is a gene listed in Table 4 other than a gene that naturally occurs in the Candida host cell.

Embodiment 96

The method of embodiment 92, wherein the first gene has at least 40 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 97

The method of embodiment 92, wherein the first gene has at least 60 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 98

The method of embodiment 92, wherein the first gene has at least 80 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 99

The method of embodiment 92, wherein the first gene has at least 95 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 100

The method of embodiment 92, wherein the first gene is encoded by a nucleic acid that binds under conditions of high stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 101

The method of embodiment 92, wherein the first gene is encoded by a nucleic acid that binds under conditions of moderate stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 102

The method of embodiment 92, wherein the first gene is encoded by a nucleic acid that binds under conditions of low stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 103

The method of embodiment 71, wherein the culture medium further comprises the substrate.

Embodiment 104

The method of embodiment 71, wherein the method further comprises making one or more second genetic modifications in a second genetic modification class to the Candida host cell, wherein the second genetic modification class comprises one or more genetic modifications that collectively or individually disrupt

-   -   the β-oxidation pathway; or     -   a gene selected from the group consisting of a CYP52A type         cytochrome P450 and a fatty alcohol oxidase.

Embodiment 105

A method for producing a genetically modified Candida host cell for the biotransformation of a substrate to a product, the method comprising making one or more genetic modifications that disrupt an alcohol dehydrogenase gene in a Candida host cell.

Embodiment 106

The method of embodiment 105, wherein the Candida host cell is Candida glabrata, Candida zeylenoides, Candida lipolytica, Candida guillermondii, Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bituminiphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida floricola, Candida floris, Candida flosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida maris, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, or Candida zeylanoides.

Embodiment 107

The method of embodiment 105, wherein the Candida host cell is Candida tropicalis.

Embodiment 108

The method of embodiment 105, wherein the Candida host cell is selected from the group consisting of DP428, DP522 and DP 527.

Embodiment 109

The method of embodiment 105, wherein the Candida host cell is genetically modified Candida tropicalis and wherein the alcohol dehydrogenase gene is selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11, and ADH-B11B.

Embodiment 110

The method of embodiment 105, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 111

The method of embodiment 105, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 112

The method of embodiment 105, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 113

The method of embodiment 105, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has at least 90 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 114

The method of embodiment 113, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 115

The method of embodiment 113, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 116

The method of embodiment 113, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 117

The method of embodiment 105, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has 100 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 118

The method of embodiment 117, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 119

The method of embodiment 117, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 120

The method of embodiment 117, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 121

The method of embodiment 105, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least one peptide selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 122

The method of embodiment 105, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least two peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 123

The method of embodiment 105, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least three peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 124

The method of embodiment 105, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least four peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 125

The method of embodiment 105, wherein the one or more genetic modifications cause an alcohol dehydrogenase to have decreased function relative to the function of the wild-type counterpart, be nonfunctional, or have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.

Embodiment 126

The method of embodiment 105 that further comprises a second genetic modification class, wherein the second genetic modification class comprises an insertion of a first gene into the Candida host cell genome; wherein the first gene encodes

-   -   a protein that is not identical to a naturally occurring protein         in the Candida host cell, or     -   a protein that is identical to a naturally occurring protein in         the Candida host cell, but expression of the gene is controlled         by a promoter that is different from the promoter that controls         the expression of the naturally occurring protein.

Embodiment 127

The method of embodiment 126, wherein the first gene encodes a desaturase, a lipase, a fatty alcohol oxidase, an alcohol dehydrogenase, a glycosyl transferase, a cytochrome P450, a cellulose, an exoglucanase, a cellobiohydrolase, an endoglucanase, a β-glucosidase, an α-amylase, a β-amylase, a γ-amylases, a glucoamylase, a maltogenase, a pullanase, an endo-β-xylanase, an α-glucuronidase, an α-arabinofuranosidase, a β-xylosidase, a β-mannanase, a β-mannosidase, a pectin lyase, an endo-polygalacturonase, an α-arabinofuranosidase, an α-galactosidase, a polymethylgalacturonase, a pectin depolymerase, a pectinase, an exopolygalacturanosidase hydrolase, an α-L-Rhamnosidase, an α-L-Arabinofuranosidase, a polymethylgalacturonate lyase, a polygalacturonate lyase, an exopolygalacturonate lyase, a peroxidase, a copper radical oxidase, an FAD-dependent oxidase, a multicopper oxidase, a lignin peroxidase or a manganese peroxidase that is

-   -   not identical to a naturally occurring protein in the Candida         host cell; or     -   identical to a naturally occurring protein in the Candida host         cell, but expression of the gene is controlled by a promoter         that is different from the promoter that controls the expression         of the naturally occurring protein.

Embodiment 128

The method of embodiment 126, wherein the first gene encodes a cytochrome P450 that is not identical to a naturally occurring cytochrome P450 in the Candida host cell.

Embodiment 129

The method of embodiment 126, wherein the first gene is a gene listed in Table 4 other than a gene that naturally occurs in the Candida host cell.

Embodiment 130

The method of embodiment 126, wherein the first gene has at least 40 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 131

The method of embodiment 126, wherein the first gene has at least 60 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 132

The method of embodiment 126, wherein the first gene has at least 80 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 133

The method of embodiment 126, wherein the first gene has at least 95 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 134

The method of embodiment 126, wherein the first gene is encoded by a nucleic acid that binds under conditions of high stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 135

The method of embodiment 126, wherein the first gene is encoded by a nucleic acid that binds under conditions of moderate stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 136

The method of embodiment 126, wherein the first gene is encoded by a nucleic acid that binds under conditions of low stringency to a nucleic acid that encodes a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 137

The method of embodiment 105, wherein the method further comprises making one or more second genetic modifications in a second genetic modification class to the Candida host cell, wherein the second genetic modification class comprises one or more genetic modifications that collectively or individually disrupt

-   -   the β-oxidation pathway; or     -   a gene selected from the group consisting of a CYP52A type         cytochrome P450 and a fatty alcohol oxidase.

Embodiment 138

A genetically modified Candida cell for the biotransformation of a substrate to a product, wherein the genetically modified Candida cell is characterized by an insertion of a first gene into the Candida host cell genome, wherein the expression of the first gene is controlled by an isocitrate lyase promoter.

Embodiment 139

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter is encoded by a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 161.

Embodiment 140

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter is encoded by a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 161.

Embodiment 141

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter is encoded by a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 161.

Embodiment 142

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter is encoded by a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 171.

Embodiment 143

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter is encoded by a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 171.

Embodiment 144

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter is encoded by a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 171.

Embodiment 145

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter comprises a sequence that has at least 60 percent sequence identity to SEQ ID NO: 161.

Embodiment 146

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter comprises a sequence that has at least 80 percent sequence identity to SEQ ID NO: 161.

Embodiment 147

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter comprises a sequence that has at least 95 percent sequence identity to SEQ ID NO: 161.

Embodiment 148

The genetically modified Candida cell of embodiment 138, wherein the isocitrate lyase promoter comprises a sequence that is identical to SEQ ID NO: 161.

Embodiment 149

The genetically modified Candida cell of embodiment 138, wherein the first gene encodes a desaturase, a lipase, a fatty alcohol oxidase, an alcohol dehydrogenase, a glycosyl transferase, a cytochrome P450, a cellulose, an exoglucanase, a cellobiohydrolase, an endoglucanase, a β-glucosidase, an α-amylase, a β-amylase, a γ-amylases, a glucoamylase, a maltogenase, a pullanase, an endo-β-xylanase, an α-glucuronidase, an α-arabinofuranosidase, a β-xylosidase, a β-mannanase, a β-mannosidase, a pectin lyase, an endo-polygalacturonase, an α-arabinofuranosidase, an α-galactosidase, a polymethylgalacturonase, a pectin depolymerase, a pectinase, an exopolygalacturanosidase hydrolase, an α-L-Rhamnosidase, an α-L-Arabinofuranosidase, a polymethylgalacturonate lyase, a polygalacturonate lyase, an exopolygalacturonate lyase, a peroxidase, a copper radical oxidase, an FAD-dependent oxidase, a multicopper oxidase, a lignin peroxidase or a manganese peroxidase.

Embodiment 150

The genetically modified Candida cell of embodiment 138, wherein the first gene encodes a cytochrome P450 that is not identical to a naturally occurring cytochrome P450 in the Candida host cell.

Embodiment 151

The genetically modified Candida cell of embodiment 138, wherein the first gene is a gene listed in Table 4 other than a gene that naturally occurs in the Candida host cell.

Embodiment 152

The genetically modified Candida cell of embodiment 138, wherein the first gene has at least 40 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 153

The genetically modified Candida cell of embodiment 138, wherein the first gene has at least 60 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 154

The genetically modified Candida cell of embodiment 138, wherein the first gene has at least 80 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 155

The genetically modified Candida cell of embodiment 138, wherein the first gene has at least 95 percent sequence identity to a gene listed in Table 4, and wherein the first gene does not naturally occur in the Candida host cell.

Embodiment 156

The genetically modified Candida cell of embodiment 138, wherein the genetically modified Candida cell is genetically modified Candida glabrata, Candida zeylenoides, Candida lipolytica, Candida guillermondii, Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bituminiphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida fioricola, Candida fioris, Candida fiosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida marts, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, or Candida zeylanoides.

Embodiment 157

The genetically modified Candida cell of embodiment 138, wherein the genetically modified Candida cell is genetically modified Candida tropicalis.

Embodiment 158

The genetically modified Candida cell of embodiment 138, wherein the genetically modified Candida cell is selected from the group consisting of DP428, DP522 and DP 527.

Embodiment 159

The genetically modified Candida cell of embodiment 138, wherein the genetically modified Candida cell further comprises one or more genetic modifications that collectively or individually disrupt an alcohol dehydrogenase gene.

Embodiment 160

The genetically modified Candida cell of embodiment 159, wherein the genetically modified Candida cell is genetically modified Candida tropicalis and wherein the alcohol dehydrogenase gene is selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11, and ADH-B11B.

Embodiment 161

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 162

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 163

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56.

Embodiment 164

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has at least 90 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 165

The genetically modified Candida cell of embodiment 164, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 166

The genetically modified Candida cell of embodiment 164, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 167

The genetically modified Candida cell of embodiment 164, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 168

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that has 100 percent sequence identity to a stretch of at least 100 contiguous residues of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO:155.

Embodiment 169

The genetically modified Candida cell of embodiment 168, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of high stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 170

The genetically modified Candida cell of embodiment 168, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of moderate stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 171

The genetically modified Candida cell of embodiment 168, wherein the alcohol dehydrogenase gene comprises a nucleic acid sequence that binds under conditions of low stringency to a first sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 56.

Embodiment 172

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least one peptide selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 173

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least two peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 174

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least three peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 175

The genetically modified Candida cell of embodiment 159, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least four peptides selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and SEQ ID NO: 160.

Embodiment 176

The genetically modified Candida cell of embodiment 159, wherein the one or more genetic modifications cause an alcohol dehydrogenase to have decreased function relative to the function of the wild-type counterpart, be nonfunctional, or have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.

Embodiment 177

A method for the biotransformation of a substrate to a product in a Candida host cell, the method comprising

-   -   inserting a first gene into the Candida host cell genome,         wherein the expression of the first gene is controlled by an         isocitrate lyase promoter; and     -   transforming the substrate to the product by fermenting the         Candida host cell in a culture medium comprising a nitrogen         source and a carbon source.

Embodiment 178

The method of embodiment 177, wherein the culture medium further comprises the substrate.

Embodiment 179

The method of embodiment 177, wherein the isocitrate lyase promoter comprises a sequence that has at least 90 percent sequence identity to SEQ ID NO: 161.

Embodiment 180

The method of embodiment 177, wherein inserting the first gene into the host Candida cell genome comprises cloning said first gene into a vector, wherein the vector comprises a stretch of at least 100 contiguous nucleotides of any one of SEQ ID NO: 171, SEQ ID NO: 161 or SEQ ID NO: 62.

Embodiment 181

The method of embodiment 177, wherein inserting the first gene into the host Candida cell genome comprises cloning said first gene into a vector, wherein the vector comprises SEQ ID NO: 161.

Embodiment 182

The method of embodiment 177, wherein inserting the first gene into the host Candida cell genome comprises cloning said first gene into a vector, wherein the vector comprises a sequence that is at least 95% identical to SEQ ID NO: 161.

Embodiment 183

A vector for an insertion of a first gene into a host Candida cell genome, wherein the first gene is under the control of an isocitrate lyase promoter in the host Candida cell genome; wherein the vector comprises a stretch of at least 100 contiguous nucleotides of any one of SEQ ID NO: 171, SEQ ID NO: 161 or SEQ ID NO: 62.

Embodiment 184

The substantially pure Candida host cell of embodiment 1, wherein said one or more genetic modifications comprise an insertion of one or more nucleic acids into the alcohol dehydrogenase gene.

Embodiment 185

The substantially pure Candida host cell of embodiment 1, wherein said one or more genetic modifications comprise a deletion of one or more nucleic acids from the alcohol dehydrogenase gene. 

What is claimed is:
 1. An α-carboxyl-ω-hydroxyl fatty acid oxidized from a substantially pure Candida host cell, wherein the substantially pure Candida host cell is characterized by: a first genetic modification class that comprises one or more genetic modifications that collectively or individually disrupt an alcohol dehydrogenase gene, and a second genetic modification class, wherein the second genetic modification class comprises an insertion of a first gene into the Candida host cell genome.
 2. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, wherein the first gene encodes a first protein that is not identical to a naturally occurring protein in the substantially pure Candida host cell, wherein the first protein oxidizes the fatty acid to the corresponding α-carboxyl-ω-hydroxyl fatty acid, or a second protein that is identical to a naturally occurring protein in the substantially pure Candida host cell, wherein the first gene encodes the second protein, expression of the first gene is controlled by a promoter that is different from a promoter that controls the expression of the naturally occurring protein, and wherein the second protein oxidizes the fatty acid to the corresponding α-carboxyl-ω-hydroxyl fatty acid.
 3. The α-carboxyl-ω-hydroxyl fatty acid of claim 2, wherein the substantially pure Candida host cell is genetically modified Candida glabrata, Candida zeylenoides, Candida lipolytica, Candida guillermondii, Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis, Candida anglica, Candida anneliseae, Candida antarctica, Candida antillancae, Candida anutae, Candida apicola, Candida apis, Candida arabinofermentans, Candida arcana, Candida ascalaphidarum, Candida asparagi, Candida atakaporum, Candida atbi, Candida athensensis, Candida atlantica, Candida atmosphaerica, Candida auringiensis, Candida auris, Candida aurita, Candida austromarina, Candida azyma, Candida azymoides, Candida barrocoloradensis, Candida batistae, Candida beechii, Candida bentonensis, Candida bertae, Candida berthetii, Candida bituminiphila, Candida blankii, Candida blattae, Candida blattariae, Candida bohiensis, Candida boidinii, Candida bokatorum, Candida boleticola, Candida bolitotheri, Candida bombi, Candida bombiphila, Candida bondarzewiae, Candida bracarensis, Candida bribrorum, Candida bromeliacearum, Candida buenavistaensis, Candida buinensis, Candida butyri, Candida californica, Candida canberraensis, Candida cariosilignicola, Candida carpophila, Candida caryicola, Candida caseinolytica, Candida castrensis, Candida catenulata, Candida cellae, Candida cellulolytica, Candida cerambycidarum, Candida chauliodes, Candida chickasaworum, Candida chilensis, Candida choctaworum, Candida chodatii, Candida chrysomelidarum, Candida cidri, Candida cloacae, Candida coipomoensis, Candida conglobata, Candida corydali, Candida cylindracea, Candida davenportii, Candida davisiana, Candida deformans, Candida dendrica, Candida dendronema, Candida derodonti, Candida diddensiae, Candida digboiensis, Candida diospyri, Candida diversa, Candida dosseyi, Candida drimydis, Candida drosophilae, Candida dubliniensis, Candida easanensis, Candida edaphicus, Candida edax, Candida elateridarum, Candida emberorum, Candida endomychidarum, Candida entomophila, Candida ergastensis, Candida ernobii, Candida etchellsii, Candida ethanolica, Candida famata, Candida fennica, Candida fermenticarens, Candida flocculosa, Candida floricola, Candida floris, Candida flosculorum, Candida fluviatilis, Candida fragi, Candida freyschussii, Candida friedrichii, Candida frijolesensis, Candida fructus, Candida fukazawae, Candida fungicola, Candida galacta, Candida galis, Candida galli, Candida gatunensis, Candida gelsemii, Candida geochares, Candida germanica, Candida ghanaensis, Candida gigantensis, Candida glaebosa, Candida glucosophila, Candida glycerinogenes, Candida gorgasii, Candida gotoi, Candida gropengiesseri, Candida guaymorum, Candida haemulonii, Candida halonitratophila, Candida halophila, Candida hasegawae, Candida hawaiiana, Candida heliconiae, Candida hispaniensis, Candida homilentoma, Candida humicola, Candida humilis, Candida hungarica, Candida hyderabadensis, Candida incommunis, Candida inconspicua, Candida insectalens, Candida insectamans, Candida insectorum, Candida intermedia, Candida ipomoeae, Candida ishiwadae, Candida jaroonii, Candida jeffriesii, Candida kanchanaburiensis, Candida karawaiewii, Candida kashinagacola, Candida kazuoi, Candida khmerensis, Candida kipukae, Candida kofuensis, Candida krabiensis, Candida kruisii, Candida kunorum, Candida labiduridarum, Candida lactis-condensi, Candida lassenensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida marls, Candida maritima, Candida maxii, Candida melibiosica, Candida membranifaciens, Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palmioleophila, Candida paludigena, Candida panamensis, Candida panamericana, Candida parapsilosis, Candida pararugosa, Candida pattaniensis, Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponderosae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida pseudovanderkliftii, Candida psychrophila, Candida pyralidae, Candida qinlingensis, Candida quercitrusa, Candida quercuum, Candida railenensis, Candida ralunensis, Candida rancensis, Candida restingae, Candida rhagii, Candida riodocensis, Candida rugopelliculosa, Candida rugosa, Candida sagamina, Candida saitoana, Candida sake, Candida salmanticensis, Candida santamariae, Candida santjacobensis, Candida saopaulonensis, Candida savonica, Candida schatavii, Candida sequanensis, Candida sergipensis, Candida shehatae, Candida silvae, Candida silvanorum, Candida silvatica, Candida silvicola, Candida silvicultrix, Candida sinolaborantium, Candida sithepensis, Candida smithsonii, Candida sojae, Candida solani, Candida songkhlaensis, Candida sonorensis, Candida sophiae-reginae, Candida sorbophila, Candida sorbosivorans, Candida sorboxylosa, Candida spandovensis, Candida steatolytica, Candida stellata, Candida stellimalicola, Candida stri, Candida subhashii, Candida succiphila, Candida suecica, Candida suzukii, Candida takamatsuzukensis, Candida taliae, Candida tammaniensis, Candida tanzawaensis, Candida tartarivorans, Candida temnochilae, Candida tenuis, Candida tepae, Candida terraborum, Candida tetrigidarum, Candida thaimueangensis, Candida thermophila, Candida tilneyi, Candida tolerans, Candida torresii, Candida tritomae, Candida tropicalis, Candida trypodendroni, Candida tsuchiyae, Candida tumulicola, Candida ubatubensis, Candida ulmi, Candida vaccinii, Candida valdiviana, Candida vanderkliftii, Candida vanderwaltii, Candida vartiovaarae, Candida versatilis, Candida vini, Candida viswanathii, Candida wickerhamii, Candida wounanorum, Candida wyomingensis, Candida xylopsoci, Candida yuchorum, Candida zemplinina, or Candida zeylanoides.
 4. The α-carboxyl-ω-hydroxyl fatty acid of claim 3, wherein the substantially pure Candida host cell is genetically modified Candida tropicalis.
 5. The α-carboxyl-ω-hydroxyl fatty acid of claim 4, wherein the substantially pure Candida host cell is genetically modified Candida tropicalis and wherein the alcohol dehydrogenase is selected from the group consisting of ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-A10, ADH-A10B, ADH-B11, and ADH-B11B.
 6. The α-carboxyl-ω-hydroxyl fatty acid of claim 5, wherein the alcohol dehydrogenase gene encodes an amino acid sequence that comprises at least three peptides selected from the group consisting of SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160.
 7. The α-carboxyl-ω-hydroxyl fatty acid of claim 2, wherein the first gene encodes a desaturase, a lipase, a fatty alcohol oxidase, an alcohol dehydrogenase, a glycosyl transferase, a cytochrome P450, a cellulose, an exoglucanase, a cellobiohydrolase, an endoglucanase, a β-glucosidase, an α-amylase, a β-amylase, a γ-amylases, a glucoamylase, a maltogenase, a pullanase, an endo-β-xylanase, an α-glucuronidase, an α-arabinofuranosidase, a β-xylosidase, a β-mannanase, a β-mannosidase, a pectin lyase, an endo-polygalacturonase, an α-arabinofuranosidase, an β-galactosidase, a polymethylgalacturonase, a pectin depolymerase, a pectinase, an exopolygalacturanosidase hydrolase, an α-L-Rhamnosidase, an α-L-Arabinofuranosidase, a polymethylgalacturonate lyase, a polygalacturonate lyase, an exopolygalacturonate lyase, a peroxidase, a copper radical oxidase, an FAD-dependent oxidase, a multicopper oxidase, a lignin peroxidase or a manganese peroxidase that is not identical to a naturally occurring protein in the substantially pure Candida host cell; or identical to a naturally occurring protein in the substantially pure Candida host cell, but expression of the gene is controlled by a promoter that is different from the promoter that controls the expression of the naturally occurring protein.
 8. The α-carboxyl-ω-hydroxyl fatty acid of claim 7, wherein the first gene encodes a cytochrome P450 that is not identical to a naturally occurring cytochrome P450 in the substantially pure Candida host cell.
 9. The α-carboxyl-ω-hydroxyl fatty acid of claim 7, wherein the first gene is a gene listed in Table 4 other than a gene that naturally occurs in the substantially pure Candida host cell.
 10. The α-carboxyl-ω-hydroxyl fatty acid of claim 6, wherein the promoter is an isocitrate lyase promoter, a cytochrome P450 promoter, a fatty alcohol oxidase promoter or an alcohol dehydrogenase promoter in the Candida host cell genome.
 11. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, further comprising a third genetic modification class, wherein the third genetic modification class comprises one or more genetic modifications in the Candida host cell genome that collectively or individually disrupt the β-oxidation pathway; or a gene selected from the group consisting of a CYP52A type cytochrome P450 and a fatty alcohol oxidase.
 12. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, wherein said one or more genetic modifications comprise an insertion of one or more nucleic acids into the alcohol dehydrogenase gene.
 13. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, wherein said one or more genetic modifications comprise a deletion of one or more nucleic acids from the alcohol dehydrogenase gene.
 14. The α-carboxyl-ω-hydroxyl fatty acid of claim 10, wherein the isocitrate lyase promoter comprises a sequence that has at least 95 percent sequence identity to SEQ ID NO:
 161. 15. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, wherein the insertion of the first gene into the host Candida cell genome comprises cloning said first gene into a vector, wherein the vector comprises a stretch of at least 100 contiguous nucleotides of SEQ ID NO:
 171. 16. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, wherein the insertion of the first gene into the host Candida cell genome comprises cloning said first gene into a vector, wherein the vector comprises SEQ ID NO:
 161. 17. The α-carboxyl-ω-hydroxyl fatty acid of claim 1, wherein the insertion of the first gene into the host Candida cell genome comprises cloning said first gene into a vector, wherein the vector comprises a sequence that is at least 95% identical to SEQ ID NO:
 161. 